N-(benzyl)aminoalkylcarboxylates, phosphinates, phosphonates and tetrazoles as Edg receptor agonists

ABSTRACT

The present invention encompasses compounds of Formula (I) as well as the pharmaceutically acceptable salts and hydrates thereof. The compounds are useful for treating immune mediated diseases and conditions, such as bone marrow, organ and tissue transplant rejection. Pharmaceutical compositions and methods of use are included.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C. §371 of PCT Application No. US03/01059 filed Jan. 14, 2003, which claimspriority under 35 U.S.C. 119 to U.S. Provisional Application No.60/349,995, filed Jan. 18, 2002.

BACKGROUND OF THE INVENTION

The present invention is related to compounds that are S1P₁/Edg1receptoragonists and thus have immunosuppressive activities by producinglymphocyte sequestration in secondary lymphoid tissues. The invention isalso directed to pharmaceutical compositions containing such compoundsand methods of treatment or prevention.

Immunosuppressive agents have been shown to be useful in a wide varietyof autoimmune and chronic inflammatory diseases, including systemiclupus erythematosis, chronic rheumatoid arthritis, type I diabetesmellitus, inflammatory bowel disease, biliary cirrhosis, uveitis,multiple sclerosis and other disorders such as Crohn's disease,ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis,autoimmune myositis, Wegener's granulomatosis, ichthyosis, Gravesophthalmopathy, atopic dermatitis and asthma. They have also proveduseful as part of chemotherapeutic regimens for the treatment ofcancers, lymphomas and leukemias.

Although the underlying pathogenesis of each of these conditions may bequite different, they have in common the appearance of a variety ofautoantibodies and/or self-reactive lymphocytes. Such self-reactivitymay be due, in part, to a loss of the homeostatic controls under whichthe normal immune system operates. Similarly, following a bone-marrow oran organ transplantation, the host lymphocytes recognize the foreigntissue antigens and begin to produce both cellular and humoral responsesincluding antibodies, cytokines and cytotoxic lymphocytes which lead tograft rejection.

One end result of an autoimmune or a rejection process is tissuedestruction caused by inflammatory cells and the mediators they release.Anti-inflammatory agents such as NSAIDs act principally by blocking theeffect or secretion of these mediators but do nothing to modify theimmunologic basis of the disease. On the other hand, cytotoxic agents,such as cyclophosphamide, act in such a nonspecific fashion that boththe normal and autoimmune responses are shut off. Indeed, patientstreated with such nonspecific immunosuppressive agents are as likely tosuccumb to infection as they are to their autoimmune disease.

Cyclosporin A is a drug used to prevent rejection of transplantedorgans. FK-506 is another drug approved for the prevention of transplantorgan rejection, and in particular, liver transplantation. Cyclosporin Aand FK-506 act by inhibiting the body's immune system from mobilizingits vast arsenal of natural protecting agents to reject the transplant'sforeign protein. Cyclosporin A was approved for the treatment of severepsoriasis and has been approved by European regulatory agencies for thetreatment of atopic dermatitis.

Though they are effective in delaying or suppressing transplantrejection, Cyclosporin A and FK-506 are known to cause severalundesirable side effects including nephrotoxicity, neurotoxicity, andgastrointestinal discomfort. Therefore, an immunosuppressant withoutthese side effects still remains to be developed and would be highlydesirable.

The immunosuppressive compound FTY720 is a lymphocyte sequestrationagent currently in clinical trials. FTY720 is metabolized in mammals toa compound that is a potent agonist of sphingosine 1-phosphatereceptors. Agonism of sphingosine 1-phosphate receptors induces thesequestration of lymphocytes (T-cells and B-cells) in lymph nodes andPeyer's patches without lymphodepletion. Such immunosuppression isdesirable to prevent rejection after organ transplantation and in thetreatment of autoimmune disorders.

Sphingosine 1-phosphate is a bioactive sphingolipid metabolite that issecreted by hematopoietic cells and stored and released from activatedplatelets. Yatomi, Y., T. Ohmori, G. Rile, F. Kazama, H. Okamoto, T.Sano, K. Satoh, S. Kume, G. Tigyi, Y. Igarashi, and Y. Ozali. 2000.Blood. 96:3431-8. It acts as an agonist on a family of G protein-coupledreceptors to regulate cell proliferation, differentiation, survival, andmotility. Fukushima, N., I. Ishii, J. J. A. Contos, J. A. Weiner, and J.Chun. 2001. Lysophospholipid receptors. Annu. Rev. Pharmacol. Toxicol.41:507-34; Hla, T., M.-J. Lee, N. Ancellin, J. H. Paik, and M. J. Kluk.2001. Lysophospholipids—Receptor revelations. Science. 294:1875-1878;Spiegel, S., and S. Milstien. 2000. Functions of a new family ofsphingosine-1-phosphate receptors. Biochim. Biophys. Acta. 1484:107-16;Pyne, S., and N. Pyne. 2000. Sphingosine 1-phosphate signalling via theendothelial differentiation gene family of G-protein coupled receptors.Pharm. & Therapeutics. 88:115-131. Five sphingosine 1-phosphatereceptors have been identified (S1P₁, S1P₂, S1P₃, S1P₄, and S1P₅, alsoknown as endothelial differentiation genes Edg1, Edg5, Edg3, Edg6,Edg8), that have widespread cellular and tissue distribution and arewell conserved in human and rodent species (see Table). Binding to S1Preceptors elicits signal transduction through Gq-, Gi/o, G12-, G13-, andRho-dependent pathways. Ligand-induced activation of S1P₁ and S1P₃ hasbeen shown to promote angiogenesis, chemotaxis, and adherens junctionassembly through Rac- and Rho-, see Lee, M.-J., S. Thangada, K. P.Claffey; N. Ancellin, C. H. Liu, M. Kluk, M. Volpi, R. I. Sha'afi, andT. Hla. 1999. Cell. 99:301-12, whereas agonism of S1P₂ promotes neuriteretraction, see Van Brocklyn, J. R., Z. Tu, L. C. Edsall, R. R. Schmidt,and S. Spiegel. 1999. J. Biol. Chem. 274:4626-4632, and inhibitschemotaxis by blocking Rac activation, see Okamoto, H., N. Takuwa, T.Yokomizo, N. Sugimoto, S. Sakurada, H. Shigematsu, and Y. Takuwa. 2000.Mol. Cell. Biol. 20:9247-9261. S1P₄ is localized to hematopoietic cellsand tissues, see Graeler, M. H., G. Bernhardt, and M. Lipp. 1999. Curr.Top. Microbiol. Immunol. 246:131-6, whereas S1P₅ is primarily a neuronalreceptor with some expression in lymphoid tissue, see Im, D. S., C. E.Heise, N. Ancellin, B. F. O'Dowd, G. J. Shei, R. P. Heavens, M. R.Rigby, T. Hla, S. Mandala, G. McAllister, S. R. George, and K. R. Lynch.2000. J. Biol. Chem. 275:14281-6. Administration of sphingosine1-phosphate to animals induces systemic sequestration of peripheralblood lymphocytes into secondary lymphoid organs, stimulatesFGF-mediated blood vessel growth and differentiation, see Lee, et al.,supra, but also has cardiovascular effects that limit the utility ofsphingosine 1-phosphate as a therapeutic agent, see Sugiyama, A., N. N.Aye, Y. Yatomi, Y. Ozaki, and K. Hashimoto. 2000. Jpn. J. Pharmacol.82:338-342. The reduced heart rate and blood pressure measured withsphingosine 1-phosphate is associated with its non-selective, potentagonist activity on all S1P receptors.

The present invention encompasses compounds which are agonists of theS1P₁/Edg1 receptor having selectivity over the S1P₃/Edg3 receptor. AnS1P₁/Edg1 receptor selective agonist has advantages over currenttherapies and extends the therapeutic window of lymphocytessequestration agents, allowing better tolerability with higher dosingand thus improving efficacy as monotherapy.

While the main use for immunosuppressants is in treating bone marrow,organ and transplant rejection, other uses for such compounds includethe treatment of arthritis, in particular, rheumatoid arthritis, insulinand non-insulin dependent diabetes, multiple sclerosis, psoriasis,inflammatory bowel disease, Crohn's disease, lupus erythematosis and thelike.

Thus, the present invention is focused on providing immunosuppressantcompounds that are safer and more effective than prior compounds. Theseand other objects will be apparent to those of ordinary skill in the artfrom the description contained herein.

Summary of S1P receptors Coupled G Name Synonyms proteins mRNAexpression S1P₁ Edg1, LP_(B1) G_(i/o) Widely distributed, endothelialcells S1P₂ Edg5, LP_(B2), G_(i/o), G_(q), Widely distributed, vascularAGR16, H218 G_(12/13) smooth muscle cells S1P₃ Edg3, LP_(B3) G_(i/o),G_(q), Widely distributed, G_(12/13) endothelial cells S1P₄ Edg6,LP_(C1) G_(i/o) Lymphoid tissues, lymphocytic cell lines S1P₅ Edg8,LP_(B4), NRG1 G_(i/o) Brain, spleen

SUMMARY OF THE INVENTION

The present invention encompasses compounds of Formula I:

as well as the pharmaceutically acceptable salts and hydrates thereof.The compounds are useful for treating immune mediated diseases andconditions, such as bone marrow, organ and tissue transplant rejection.Pharmaceutical compositions and methods of use are included.

DETAILED DESCRIPTION OF THE INVENTION

The invention encompasses a compound of Formula I

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

-   Ar is phenyl or naphthyl;-   A is selected from: —CO₂H, 1H-tetrazol-5-yl, —PO₃H₂, —PO₂H₂, —SO₃H,    and —PO(R⁵)OH, wherein R⁵ is selected from the group consisting of:    C₁₋₄alkyl, hydroxyC₁₋₄alkyl, phenyl, —C(O)—C₁₋₃alkoxy and    —CH(OH)-phenyl, said phenyl and phenyl portion of —CH(OH)-phenyl    optionally substituted with 1-3 substituents independently selected    from the group consisting of: hydroxy, halo, —CO₂H, C₁₋₄alkyl,    —S(O)_(k)C₁₋₃alkyl, wherein k is 0, 1 or 2, C₁₋₃alkoxy, C₃₋₆    cycloalkoxy, aryl and aralkoxy, the alkyl portions of said    C₁₋₄alkyl, —S(O)_(k)C₁₋₃alkyl, C₁₋₃alkoxy and C₃₋₆ cycloalkoxy    optionally substituted with 1-3 halo groups;-   n is 2, 3 or 4;-   each R¹ and R² is each independently selected from the group    consisting of: hydrogen, halo, hydroxy, —CO₂H, C₁₋₆alkyl and phenyl,    said C₁₋₆alkyl and phenyl optionally substituted with 1-3 halo    groups;-   R³ is selected from the group consisting of: hydrogen and C₁₋₄alkyl,    optionally substituted with 1-3 hydroxy or halo groups;-   each R⁴ is independently selected from the group consisting of:    hydroxy, halo, —CO₂H, C₁₋₄alkyl, —S(O)_(k)C₁₋₃alkyl, wherein k is 0,    1 or 2, C₁₋₃alkoxy, C₃₋₆ cycloalkoxy, aryl and aralkoxy, the alkyl    portions of said C₁₋₄alkyl, —S(O)_(k)C₁₋₃alkyl, C₁₋₃alkoxy and C₃₋₆    cycloalkoxy optionally substituted with 1-3 halo groups;-   C is selected from the group consisting of:    -   (1) C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl or —CHOH—C₁₋₆alkyl,        said C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl and —CHOH—C₁₋₆alkyl        optionally substituted with phenyl, and    -   (2) phenyl or HET, each optionally substituted with 1-3        substituents independently selected from the group consisting        of: halo, phenyl, C₁₋₄alkyl and C₁₋₄alkoxy, said C₁₋₄alkyl and        C₁₋₄alkoxy groups optionally substituted from one up to the        maximum number of substitutable positions with a substituent        independently selected from halo and hydroxy, and said phenyl        optionally substituted with 1 to 5 groups independently selected        from the group consisting of: halo and C₁₋₄alkyl, optionally        substituted with 1-3 halo groups,-   or C is not present;-   when C is not present then B is selected from the group consisting    of: phenyl, C₅₋₁₆alkyl, C₅₋₁₆alkenyl, C₅₋₁₆alkynyl,    —CHOH—C₄₋₁₅alkyl, —CHOH—C₄₋₁₅alkenyl, —CHOH—C₄₋₁₅alkynyl,    C₄₋₁₅alkoxy, —O—C₄₋₁₅alkenyl, —O—C₄₋₁₅alkynyl, C₄₋₁₅alkylthio,    —S—C₄₋₁₅alkenyl, —S—C₄₋₁₅alkynyl, —CH₂—C₃₋₁₄alkoxy,    —CH₂—O—C₃₋₁₄alkenyl, —CH₂—O—C₃₋₁₄alkynyl, —(C═O)—C₄₋₁₅alkyl,    —(C═O)—C₄₋₁₅alkenyl, —(C═O)—C₄₋₁₅alkynyl, —(C═O)—O—C₃₋₁₄alkyl,    —(C═O)—O—C₃₋₁₄alkenyl, —(C═O)—O—C₃₋₁₄alkynyl,    —(C═O)—N(R⁶)(R⁷)—C₃₋₁₄alkyl, —(C═O)—N(R⁶)(R⁷)—C₃₋₁₄alkenyl,    —(C═O)—N(R⁶)(R⁷)—C₃₋₁₄alkynyl, —N(R⁶)(R⁷)—(C═O)-C₃₋₁₄alkyl,    —N(R⁶)(R⁷)—(C═O)-C₃₋₁₄alkenyl and —N(R⁶)(R⁷)—(C═O)—C₃₋₁₄alkynyl,-   when C is phenyl or HET then B is selected from the group consisting    of: C₁₋₆alkyl, C₁₋₅alkoxy, —(C═O)—C₁₋₅alkyl, —(C═O)—O—C₁₋₄alkyl,    —(C═O)—N(R⁶)(R⁷)—C₁₋₄alkyl,

phenyl and HET, and

-   when C is C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl or —CHOH—C₁₋₆alkyl    then B is phenyl; and-   R⁶ and R⁷ are independently selected from the group consisting of:    hydrogen, C₁₋₉alkyl and —(CH₂)_(p)-phenyl, wherein p is 1 to 5 and    phenyl is optionally substituted with 1-3 substituents independently    selected from the group consisting of: C₁₋₃alkyl and C₁₋₃alkoxy,    each optionally substituted with 1-3 halo groups.

For purposes of this specification, C may be substituted at anysubstitutable position on B. For example, when B is methoxy and C isthiophene, thiophene replaces a hydrogen on the methoxy group. Furthervariations are illustrated in the examples that follow. Also, the pointof any attachments shown for B is to the Ar group. For example, when Bis —(C═O)—C₆₋₁₁alkynyl this means B is attached to Ar as follows:Ar—(C═O)—C₆₋₁₁alkynyl. C may then be substituted at any substituableposition on B.

An embodiment of the invention encompasses a compound of Formula Iwherein BET is selected from the group consisting of:

For purposes of this specification HET can be attached at any point ofattachment and substituents can be substituted at any substituableposition. Such points of attachments and substituable positions areascertainable to one having ordinary skill in the art.

An embodiment of the invention encompasses a compound of Formula Iwherein n is 2.

An embodiment of the invention encompasses a compound of Formula Iwherein n is 3.

An embodiment of the invention encompasses a compound of Formula Iwherein each R¹ and R² is independently selected from the groupconsisting of: hydrogen, —CO₂H, hydroxy, halo, C₁₋₃alkyl and phenyl.

An embodiment of the invention encompasses a compound of Formula Iwherein A is PO₃H₂.

An embodiment of the invention encompasses a compound of Formula Iwherein A is —CO₂H.

An embodiment of the invention encompasses a compound of Formula Iwherein A is PO(R⁵)OH, wherein R⁵ is selected from the group consistingof: C₁₋₄alkyl, hydroxyC₁₋₄alkyl, C(O)—C₁₋₂alkoxy and benzyl, whereinboth the methyl and phenyl portions of said benzyl are optionallysubstituted with 1-3 halo or hydroxy groups.

An embodiment of the invention encompasses a compound of Formula Iwherein A is PO₂H₂.

An embodiment of the invention encompasses a compound of Formula Iwherein A is 1H-tetrazol-5-yl.

An embodiment of the invention encompasses a compound of Formula Iwherein R³ is hydrogen or methyl.

An embodiment of the invention encompasses a compound of Formula Iwherein each R⁴ is independently selected from the group consisting of:halo, hydroxy, C1-3alkyl, C1-3alkoxy, C1-3alkylthio, phenyl, benzyloxyand cyclopropyloxy.

An embodiment of the invention encompasses a compound of Formula Iwherein B is C₈₋₁₀alkyl and C is not present.

An embodiment of the invention encompasses a compound of Formula Iwherein B is C₄₋₁₁alkoxy and C is not present.

An embodiment of the invention encompasses a compound of Formula Iwherein B is phenyl, optionally substituted with 1-3 substituentsindependently selected from the group consisting of: halo, C₁₋₄alkyl andC₁₋₄alkoxy, and C is selected from the group consisting of: hydrogen,phenyl, C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl and

—CHOH—C₁₋₆alkyl, said C₁₋₈alkyl, C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl and—CHOH—C₁₋₆alkyl optionally substituted with phenyl.

An embodiment of the invention encompasses a compound of Formula Iwherein B is selected from the group consisting of: —CHOH—C₆₋₁₀alkyl,C₆₋₁₀alkylthio, —CH₂—C₅₋₉alkoxy, —(C═O)—C₆₋₁₀alkyl, —(C═O)—O—C₅₋₉alkyl,—(C═O)—N(R⁶)(R⁷)—C₅₋₉alkyl, —N(R⁶)(R⁷)—(C═O)—C₅₋₉alkyl, and C is notpresent.

An embodiment of the invention encompasses a compound of Formula Iwherein B is C₁₋₆alkyl or C₁₋₅alkoxy and C is phenyl.

An embodiment of the invention encompasses a compound of Formula Iwherein B—C is

An embodiment of the invention encompasses a compound of Formula Iwherein Ar is phenyl and the group —B—C is attached to the phenyl ringat the 3- or 4-position.

An embodiment of the invention encompasses a compound of Formula II

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   the group —B—C is attached to the phenyl ring at the 3- or    4-position;-   n is 2, 3 or 4;-   each R¹ and R² is independently selected from the group consisting    of: hydrogen, —CO₂H, hydroxy, halo, C₁₋₃alkyl and phenyl, said    C₁₋₃alkyl and phenyl optionally substituted with 1-3 halo group;-   A is selected from the group consisting of: 1H-tetrazol-5-yl, PO₂H₂,    PO₃H₂, —CO₂H and PO(R⁵)OH, wherein R⁵ is selected from the group    consisting of: C₁₋₄alkyl, hydroxyC₁₋₄alkyl, C(O)—C₁₋₂alkoxy and    benzyl, wherein both the methyl and phenyl portions of said benzyl    are optionally substituted with 1-3 halo or hydroxy groups;-   R³ is hydrogen or methyl;-   each R⁴ is independently selected from the group consisting of:    halo, hydroxy, C₁₋₃alkyl, C₁₋₃alkoxy, C₁₋₃alkylthio, phenyl,    benzyloxy and cyclopropyloxy; and-   B—C is selected from the group consisting of:    -   (1) B is C₈₋₁₀alkyl and C is not present.    -   (2) B is C₄₋₁₁alkoxy and C is not present.    -   (3) B is phenyl, optionally substituted with 1-3 substituents        independently selected from the group consisting of: halo,        C₁₋₄alkyl and C₁₋₄alkoxy, and C is selected from the group        consisting of: hydrogen, phenyl, C₁₋₈alkyl, C₁₋₈alkoxy        —(C═O)—C₁₋₆alkyl and —CHOH—C₁₋₆alkyl, said C₁₋₈alkyl,        C₁₋₈alkoxy, —(C═O)—C₁₋₆alkyl and —CHOH—C₁₋₆alkyl optionally        substituted with phenyl;    -   (4) B is —CHOH—C₆₋₁₀alkyl, C₆₋₁₀alkylthio, —CH₂—C₅₋₉alkoxy,        —(C═O)—C₆₋₁₀alkyl, —(C═O)—O—C₅₋₉alkyl,        —(C═O)—N(R⁶)(R⁷)—C₅₋₉alkyl or —N(R⁶)(R⁷)—(C═O)—C₅₋₉alkyl, and C        is not present.    -   (5) B is C₁₋₆alkyl or C₁₋₅alkoxy and C is phenyl.    -   (6) B—C is

The invention also encompasses a method of treating an immunoregulatoryabnormality in a mammalian patient in need of such treatment comprisingadministering to said patient a compound of Formula I in an amount thatis effective for treating said immunoregulatory abnormality.

Within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is an autoimmune or chronic inflammatorydisease selected from the group consisting of: systemic lupuserythematosis, chronic rheumatoid arthritis, type I diabetes mellitus,inflammatory bowel disease, biliary cirrhosis, uveitis, multiplesclerosis, Crohn's disease, ulcerative colitis, bullous pemphigoid,sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis,ichthyosis, Graves ophthalmopathy and asthma.

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is bone marrow or organ transplantrejection or graft-versus-host disease.

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is selected from the group consisting of:transplantation of organs or tissue, graft-versus-host diseases broughtabout by transplantation, autoimmune syndromes including rheumatoidarthritis, systemic lupus erythematosus, Hashimoto's thyroiditis,multiple sclerosis, myasthenia gravis, type I diabetes, uveitis,posterior uveitis, allergic encephalomyelitis, glomerulonephritis,post-infectious autoimmune diseases including rheumatic fever andpost-infectious glomerulonephritis, inflammatory and hyperproliferativeskin diseases, psoriasis, atopic dermatitis, contact dermatitis,eczematous dermatitis, seborrhoeic dermatitis, lichen planus, pemphigus,bullous pemphigoid, epidermolysis bullosa, urticaria, angioedemas,vasculitis, erythema, cutaneous eosinophilia, lupus erythematosus, acne,alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitisassociated with Behcet's disease, keratitis, herpetic keratitis, conicalcornea, dystrophia epithelialis corneae, corneal leukoma, ocularpemphigus, Mooren's ulcer, scleritis, Graves' opthalmopathy,Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergies, reversibleobstructive airway disease, bronchial asthma, allergic asthma, intrinsicasthma, extrinsic asthma, dust asthma, chronic or inveterate asthma,late asthma and airway hyper-responsiveness, bronchitis, gastric ulcers,vascular damage caused by ischemic diseases and thrombosis, ischemicbowel diseases, inflammatory bowel diseases, necrotizing enterocolitis,intestinal lesions associated with thermal burns, coeliac diseases,proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease,ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischernic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, and chronic bacterialinfection

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is multiple sclerosis

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is rheumatoid arthritis

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is systemic lupus erythematosus

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is psoriasis

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is rejection of transplanted organ ortissue

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is inflammatory bowel disease.

Also within this embodiment is encompassed the above method wherein theimmunoregulatory abnormality is a malignancy of lymphoid originincluding acute and chronic lymphocytic leukemias and lymphomas.

The invention also encompasses a method of suppressing the immune systemin a mammalian patient in need of immunosuppression comprisingadministering to said patient an immunosuppressing effective amount of acompound of Formula I.

The invention also encompasses a pharmaceutical composition comprised ofa compound of Formula I in combination with a pharmaceuticallyacceptable carrier.

Exemplifying the invention are the following compounds:

Example Number Structure 1

2

3

4

5

6

7

8

9

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

The invention is described using the following definitions unlessotherwise indicated.

The term “halogen” or “halo” includes F, Cl, Br, and I.

The term “alkyl” means linear or branched structures and combinationsthereof, having the indicated number of carbon atoms. Thus, for example,C₁₋₆alkyl includes methyl, ethyl, propyl, 2-propyl, s- and t-butyl,butyl, pentyl, hexyl, 1,1-dimethylethyl, cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

The term “alkoxy” means alkoxy groups of a straight, branched or cyclicconfiguration having the indicated number of carbon atoms. C₁₋₆alkoxy,for example, includes methoxy, ethoxy, propoxy, isopropoxy, and thelike.

The term “alkylthio” means alkylthio groups having the indicated numberof carbon atoms of a straight, branched or cyclic configuration.C₁₋₆alkylthio, for example, includes methylthio, propylthio,isopropylthio, and the like.

The term “alkenyl” means linear or branched structures and combinationsthereof, of the indicated number of carbon atoms, having at least onecarbon-to-carbon double bond, wherein hydrogen may be replaced by anadditional carbon-to-carbon double bond. C₂₋₆alkenyl, for example,includes ethenyl, propenyl, 1-methylethenyl, butenyl and the like.

The term “alkynyl” means linear or branched structures and combinationsthereof, of the indicated number of carbon atoms, having at least onecarbon-to-carbon triple bond. C₃₋₆allynyl, for example, includes,propenyl, 1-methylethenyl, butenyl and the like.

The term “cycloalkyl” means mono-, bi- or tri-cyclic structures,optionally combined with linear or branched structures, the indicatednumber of carbon atoms. Examples of cycloalkyl groups includecyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl,2-ethyl-1-bicyclo[4.4.0]decyl, and the like.

The term “aryl” is defined as a mono- or bi-cyclic aromatic ring systemand includes, for example, phenyl, naphthyl, and the like.

The term “aralkyl” means an alkyl group as defined above of 1 to 6carbon atoms with an aryl group as defined above substituted for one ofthe alkyl hydrogen atoms, for example, benzyl and the like.

The term “aryloxy” means an aryl group as defined above attached to amolecule by an oxygen atom (aryl-O) and includes, for example, phenoxy,naphthoxy and the like.

The term “aralkoxy” means an aralkyl group as defined above attached toa molecule by an oxygen atom (aralkyl-O) and includes, for example,benzyloxy, and the like.

The term “arylthio” is defined as an aryl group as defined aboveattached to a molecule by an sulfur atom (aryl-S) and includes, forexample, thiophenyoxy, thionaphthoxy and the like.

The term “aroyl” means an aryl group as defined above attached to amolecule by an carbonyl group (aryl-C(O)—) and includes, for example,benzoyl, naphthoyl and the like.

The term “aroyloxy” means an aroyl group as defined above attached to amolecule by an oxygen atom (aroyl-O) and includes, for example,benzoyloxy or benzoxy, naphthoyloxy and the like.

The term “BET” is defined as a 5- to 10-membered aromatic, partiallyaromatic or non-aromatic mono- or bicyclic ring, containing 1-5heteroatoms selected from O, S and N, and optionally substituted with1-2 oxo groups. Preferably, “HET” is a 5- or 6-membered aromatic ornon-aromatic monocyclic ring containing 1-3 heteroatoms selected from O,S and N, for example, pyridine, pyrimidine, pyridazine, furan,thiophene, thiazole, oxazole, isooxazole and the like, or heterocycle isa 9- or 10-membered aromatic or partially aromatic bicyclic ringcontaining 1-3 heteroatoms selected from O, S, and N, for example,benzofuran, benzothiophene, indole, pyranopyrrole, benzopyran,quionoline, benzocyclohexyl, naphtyridine and the like. “ET” alsoincludes the following: benzimidazolyl, benzofuranyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl,indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl,pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl. Apreferred group of HET is as follows:

The term “treating” encompasses not only treating a patient to relievethe patient of the signs and symptoms of the disease or condition butalso prophylactically treating an asymptomatic patient to prevent theonset or progression of the disease or condition. The term “amounteffective for treating” is intended to mean that amount of a drug orpharmaceutical agent that will elicit the biological or medical responseof a tissue, a system, animal or human that is being sought by aresearcher, veterinarian, medical doctor or other clinician. The termalso encompasses the amount of a pharmaceutical drug that will preventor reduce the risk of occurrence of the biological or medical event thatis sought to be prevented in a tissue, a system, animal or human by aresearcher, veterinarian, medical doctor or other clinician.

The invention described herein includes pharmaceutically acceptablesalts and hydrates. Pharmaceutically acceptable salts include both themetallic (inorganic) salts and organic salts; a list of which is givenin Remington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985).It is well known to one skilled in the art that an appropriate salt formis chosen based on physical and chemical stability, flowability,hydroscopicity and solubility. As will be understood by those skilled inthe art, pharmaceutically acceptable salts include, but are not limitedto salts of inorganic acids such as hydrochloride, sulfate, phosphate,diphosphate, hydrobromide, and nitrate or salts of an organic acid suchas malate, maleate, fumarate, tartrate, succinate, citrate, acetate,lactate, methanesulfonate, p-toluenesulfonate or pamoate, salicylate andstearate. Similarly pharmaceutically acceptable cations include, but arenot limited to sodium, potassium, calcium, aluminum, lithium andammonium (especially ammonium salts with secondary amines). Preferredsalts of this invention for the reasons cited above include potassium,sodium, calcium and ammonium salts. Also included within the scope ofthis invention are crystal forms, hydrates and solvates of the compoundsof Formula I.

For purposes of this Specification, “pharmaceutically acceptablehydrate” means the compounds of the instant invention crystallized withone or more molecules of water to form a hydrated form.

The invention also includes the compounds falling within formula I inthe form of one or more stereoisomers, in substantially pure form or inthe form of a mixture of stereoisomers. All such isomers are encompassedwithin the present invention.

By virtue of their S1P₁/Edg1 agonist activity, the compounds of thepresent invention are immunoregulatory agents useful for treating orpreventing automimmune or chronic inflammatory diseases. The compoundsof the present invention are useful to suppress the immune system ininstances where immunosuppression is in order, such as in bone marrow,organ or transplant rejection, autoimmune and chronic inflammatorydiseases, including systemic lupus erythematosis, chronic rheumatoidarthritis, type I diabetes mellitus, inflammatory bowel disease, biliarycirrhosis, uveitis, multiple sclerosis, Crohn's disease, ulcerativecolitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmunemyositis, Wegener's granulomatosis, ichthyosis, Graves ophthalmopathyand asthma.

More particularly, the compounds of the present invention are useful totreat or prevent a disease or disorder selected from the groupconsisting of: transplantation of organs or tissue, graft-versus-hostdiseases brought about by transplantation, autoimmune syndromesincluding rheumatoid arthritis, systemic lupus erythematosus,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type Idiabetes, uveitis, posterior uveitis, allergic encephalomyelitis,glomerulonephritis, post-infectious autoimmune diseases includingrheumatic fever and post-infectious glomerulonephritis, inflammatory andhyperproliferative skin diseases, psoriasis, atopic dermatitis, contactdermatitis, eczematous dermatitis, seborrhoeic dermatitis, lichenplanus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria,angioedemas, vasculitis, erythema, cutaneous eosinophilia, lupuserythematosus, acne, alopecia areata, keratoconjunctivitis, vernalconjunctivitis, uveitis associated with Behcet's disease, keratitis,herpetic keratitis, conical cornea, dystrophia epithelialis corneae,corneal leukoma, ocular pemphigus, Mooren's ulcer, scleritis, Graves'opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollenallergies, reversible obstructive airway disease, bronchial asthma,allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma,chronic or inveterate asthma, late asthma and airwayhyper-responsiveness, bronchitis, gastric ulcers, vascular damage causedby ischeric diseases and thrombosis, ischemic bowel diseases,inflammatory bowel diseases, necrotizing enterocolitis, intestinallesions associated with thermal burns, coeliac diseases, proctitis,eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerativecolitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischemic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, and chronic bacterialinfection.

Also embodied within the present invention is a method of preventing ortreating resistance to transplantation or transplantation rejection oforgans or tissues in a mammalian patient in need thereof, whichcomprises administering a therapeutically effective amount of thecompound of Formula I.

A method of suppressing the immune system in a mammalian patient in needthereof, which comprises administering to the patient an immune systemsuppressing amount of the compound of Formula I is yet anotherembodiment.

Most particularly, the method described herein encompasses a method oftreating or preventing bone marrow or organ transplant rejection whichis comprised of administering to a mammalian patient in need of suchtreatment or prevention a compound of formula I, or a pharmaceuticallyacceptable salt or hydrate thereof, in an amount that is effective fortreating or preventing bone marrow or organ transplant rejection.

Furthermore, a preferred group of compounds of the present invention areagonists of the S1P₁/Edg1 receptor having selectivity over S1P₃/Edg3receptor. An Edg1 selective agonist has advantages over currenttherapies and extends the therapeutic window of lymphocytessequestration agents, allowing better tolerability with higher dosingand thus improving efficacy as monotherapy. The following compoundspossesses a selectivity for the S1P1/Edg1 receptor over the S1PR₃/Edg3receptor of at least 20 fold as measured by the ratio of EC₅₀ for theS1P₁/Edg1 receptor to the EC₅₀ for the S1P₃/Edg3 receptor as evaluatedin the ³⁵S-GTPγS binding assay and possesses an EC₅₀ for binding to theS1P₁/Edg1 receptor of 100 nM or less as evaluated by the ³⁵S-GTPγSbinding assay:

The present invention also includes a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and the compound ofFormula I or a pharmaceutically acceptable salt or hydrate thereof. Apreferred embodiment of the formulation is one where a secondimmunosuppressive agent is also included. Examples of such secondimmunosuppressive agents are, but are not limited to azathioprine,brequinar sodium, deoxyspergualin, mizaribine, mycophenolic acidmorpholino ester, cyclosporin, FK-506, rapamycin and FTY720.

The present compounds, including salts and hydrates thereof, are usefulin the treatment of autoimmune diseases, including the prevention ofrejection of bone marrow transplant, foreign organ transplants and/orrelated afflictions, diseases and illnesses.

The compounds of this invention can be administered by any means thateffects contact of the active ingredient compound with the site ofaction in the body of a warm-blooded animal. For example,administration, can be oral, topical, including transdermal, ocular,buccal, intranasal, inhalation, intravaginal, rectal, intracisternal andparenteral. The term “parenteral” as used herein refers to modes ofadministration which include subcutaneous, intravenous, intramuscular,intraarticular injection or infusion, intrastemal and intraperitoneal.

The compounds can be administered by any conventional means availablefor use in conjunction with pharmaceuticals, either as individualtherapeutic agents or in a combination of therapeutic agents. They canbe administered alone, but are generally administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage administered will be dependent on the age, health and weightof the recipient, the extent of disease, kind of concurrent treatment,if any, frequency of treatment and the nature of the effect desired.Usually, a daily dosage of active ingredient compound will be from about0.1-2000 milligrams per day. Ordinarily, from 1 to 100 milligrams perday in one or more applications is effective to obtain desired results.These dosages are the effective amounts for the treatment of autoimmunediseases, the prevention of rejection of foreign organ transplantsand/or related afflictions, diseases and illnesses.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, troches, dragées, granules and powders, or inliquid dosage forms, such as elixirs, syrups, emulsions, dispersions,and suspensions. The active ingredient can also be administeredparenterally, in sterile liquid dosage forms, such as dispersions,suspensions or solutions. Other dosages forms that can also be used toadminister the active ingredient as an ointment, cream, drops,transdermal patch or powder for topical administration, as an ophthalmicsolution or suspension formation, i.e., eye drops, for ocularadministration, as an aerosol spray or powder composition for inhalationor intranasal administration, or as a cream, ointment, spray orsuppository for rectal or vaginal administration.

Gelatin capsules contain the active ingredient and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene gycols are suitable carriers for parenteral solutions.Solutions for parenteral administration preferably contain a watersoluble salt of the active ingredient, suitable stabilizing agents, andif necessary, buffer substances. Antioxidizing agents such as sodiumbisulfite, sodium sulfite, or ascorbic acid, either alone or combined,are suitable stabilizing agents. Also used are citric acid and its saltsand sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propylparaben,and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield.

For administration by inhalation, the compounds of the present inventionmay be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or nebulisers. The compounds mayalso be delivered as powders which may be formulated and the powdercomposition may be inhaled with the aid of an insufflation powderinhaler device. The preferred delivery system for inhalation is ametered dose inhalation (MDI) aerosol, which may be formulated as asuspension or solution of a compound of Formula I in suitablepropellants, such as fluorocarbons or hydrocarbons.

For ocular administration, an ophthalmic preparation may be formulatedwith an appropriate weight percent solution or suspension of thecompounds of Formula I in an appropriate ophthalmic vehicle, such thatthe compound is maintained in contact with the ocular surface for asufficient time period to allow the compound to penetrate the cornealand internal regions of the eye.

Useful pharmaceutical dosage-forms for administration of the compoundsof this invention can be illustrated as follows:

Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 milligrams of powderedactive ingredient, 150 milligrams of lactose, 50 milligrams ofcellulose, and 6 milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil is prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 milligrams of the active ingredient. The capsules arewashed and dried.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit is 100 milligrams of active ingredient, 0.2milligrams of colloidal silicon dioxide, 5 milligrams of magnesiumstearate, 275 milligrams of microcrystalline cellulose, 11 milligrams ofstarch and 98.8 milligrams of lactose. Appropriate coatings may beapplied to increase palatability or delay absorption.

Injectable

A parenteral composition suitable for administration by injection isprepared by stirring 1.5% by weight of active ingredient in 10% byvolume propylene glycol. The solution is made to volume with water forinjection and sterilized.

Suspension

An aqueous suspension is prepared for oral administration so that each 5milliliters contain 100 milligrams of finely divided active ingredient,100 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodiumbenzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 millilitersof vanillin.

The same dosage forms can generally be used when the compounds of thisinvention are administered stepwise or in conjunction with anothertherapeutic agent. When drugs are administered in physical combination,the dosage form and administration route should be selected depending onthe compatibility of the combined drugs. Thus the term coadministrationis understood to include the administration of the two agentsconcomitantly or sequentially, or alternatively as a fixed dosecombination of the two active components.

Methods of Synthesis

Two general methods that can be employed to prepare compounds in thecurrent invention are depicted in Scheme 1. Intermediates i are in manycases available from commercial sources (e.g., β-alanine, where A=—CO₂H,R₁=H, R₂=H, n=2; 4-(amino)butanoic acid, where A=—CO₂H, R₁=H, R₂=H, n=3;3-(amino)propyl phosphonic acid, where A=—PO₃H₂, R₁=H, R₂=H, n=3).Intermediates i can also be prepared using methods known to thoseskilled in the art or using methods described below. Combining i with anaryl aldehyde ii in the presence of an appropriate reducing agent (e.g.,sodium cyanoborohydride, sodium triacetoxyborohydride, sodiumborohydride) in a compatible solvent (e.g., methanol, ethanol,acetonitrile, methylene chloride) can afford compounds of structure iii.Alternatively, intermediates i can be combined with a benzyl halide orsulfonate ester iv in the presence of an appropriate base (e.g., sodiumcarbonate, potassium carbonate, triethylamine,N,N-diisopropylethylamine) in a compatible solvent (e.g., methanol,ethanol, acetonitrile) at or above room temperature to give compounds ofstructure iii. In cases where A in structure i would interfere with thetransformation to iii, an appropriate protecting group (Greene & Wuts,eds., “Protecting Groups in Organic Synthesis”, John Wiley & Sons, Inc.)that would mask A and allow for the liberation of A after coupling witheither ii or iv can be employed. In cases where iii contains asymmetriccenters, the individual stereoisomers of iii can obtained by methodsknown to those skilled in the art which include (but are not limitedto): stereospecific synthesis, resolution of salts of iii or any of theintermediates used in its preparation with enantiopure acids or bases,resolution of iii or any of the intermediates used in its preparation byHPLC employing enantiopure stationary phases.

Methods to prepare analogs iii in which R₁=H, R₂=H, n=3 and A=—PO₂H₂ andR₁=H, R₂=H, n=3 and A=—PO(OH)R₅ are shown in Scheme 2. Ethyldiethoxymethylphosphinic acid (v) can be treated with acrylonitrile inthe presence of a base (e.g., sodium hydride, sodium ethoxide, lithiumdiisopropylamide) in a suitable solvent (e.g., EtOH, THF) at or belowroom temperature to afford vi. Reduction of the cyano group of vi usingcatalytic hydrogenation affords vii which can be converted to viii usingthe methods described in Scheme 1 to convert i to iii. Treating viiiwith strong aqueous acid at or above room temperature can give iii inwhich R₁=H, R₂=H, n=3 and A=—PO₂H₂. Phosphinic acid alkylation can becarried out by conversion of the phosphinic acid to thebis(trimethylsilyl) ester and treating it with an electrophile (e.g., analkyl halide, an alkyl or aryl aldehyde) to give the alkylated product(R₁=H, R₂=H, n=3 and A=—PO(OH)R₅).

Several methods that can be used to prepare compounds that can beemployed as intermediate ii in Scheme 1 above are shown in Scheme 3.Many aryl carboxylic acids, aryl carboxylic acid halides, arylcarboxylic esters, and aryl N-alkoxyl-N-alkyl carboxamides (ix) arecommercially available and can be converted to aryl aldehydes (x) usingreduction methods known by those skilled in the art (see Larock,“Comprehensive Organic Transformations, A Guide to Functional GroupPreparations”, VCH Publishers, Inc.). Alternatively, many benzylalcohols (xi) are commercially available and can be converted to arylaldehydes (xii) using oxidation methods known by those skilled in theart. For cases where B=alkoxy, a hydroxy benzaldehyde xiii can becombined with a alkyl halide or sulfonate ester in the presence of anappropriate base (e.g., sodium hydride, sodium carbonate, potassiumcarbonate, triethylamine, N,N-diisopropylethylamine) in a compatiblesolvent (e.g., DMF, methanol, ethanol, acetonitrile) at or above roomtemperature to give compounds of structure xiv. Alternatively, a hydroxybenzaldehyde xiii can be combined with an alcohol, a dialkylazodicarboxylate (e.g., diethyl azodicarboxylate,diisopropylazodicarboxylate) and triphenylphosphene in an appropriatesolvent C(THF, toluene, methylene chloride) to give xiv. For cases whereB is 1,2,4-oxadiazolyl, N-hydroxyamidine xv can be treated with an acidchloride in an appropriate solvent (xylenes, toluene) in the presence ofan amine base (pyridine, DBU) with heating to give an intermediate xvi.Alternatively, xv can be treated with a carboxylic acid, a carbodiimide(e.g., N,N′-dicyclohexylcarbodiimide,1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and1-hydroxybenzotriazole in an appropriate solvent (xylenes, toluene) togive xvi. Prepared by either manner, the ester group of xv can beconverted to aldehyde with methods employed to convert ix to x. Forcases where B is —(C═O)C₆₋₁₁ alkyl and R₄=H, an aryl 1,4-dialdehyde(xvii) can be treated with a limiting amount of an alkyl organometallicreagent (e.g., alkyl magnesium bromide, alkyl lithium) at or below roomtemperature in an ethereal solvent (e.g., THF, diethyl ether,1,2-dimethoxyethane) to afford intermediate xviii. Mild oxidation ofxviii (e.g., treatment with oxalyl chloride and DMSO at −78° C. indichloromethane

followed by a trialkylamine base and warming (Swern oxidation);treatment with 4-methylmorpholine N-oxide and catalytictetrapropylammonium peruthenate in acetonitrile; Dess-Martin reagent inmethylene chloride) can give aldehyde xix.

Several other methods that can be used to prepare compounds that can beemployed as intermediate ii in Scheme 1 above are shown in Scheme 4. Forintermediates in which B=phenyl and R₄=H, 4-(formyl)phenyl boronic acid(xx) can by reacted with an aryl bromide, iodide ortrifluoromethanesulfonate ester in the presence of a palladium catalyst(e.g., tetrakis(triphenylphosphine)palladium,2-(dicyclohexylphosphino)biphenyl and palladium acetate) in the presenceof an appropriate base (e.g., potassium carbonate, potassium fluoride)in an appropriate solvent (e.g., ethanol, 1,4-dioxane, THF) at or aboveroom temperature to give xxi. Intermediates in which the phenyl ring issubstituted with an amide linkage (either xxiii or xxv) can be preparedby methods known by those skilled in the art to prepare amides fromcarboxylic acid derivatives (see Larock, “Comprehensive OrganicTransformations, A Guide to Functional Group Preparations”, VCHPublishers, Inc.). Additionally, ii can be prepared by treating an arylbromide (xxvi) with an alkyl lithium (e.g., n-butyllithium,t-butyllithium) in a compatible solvent (e.g., diethyl ether,1,2-dimethoxyethane, THF) at or below room temperature followed byreacting the formed aryl lithium with N,N-dimethylformamide to give ii.

Methods for preparing the compounds of this invention are furtherillustrated in the following examples. Alternative routes will be easilydiscernible to practitioners in the field.

General Methods

Concentration of solutions was carried out on a rotary evaporator underreduced pressure. Conventional flash chromatography was carried out onsilica gel (230-400 mesh). Flash chromatography was also carried outusing a Biotage Flash Chromatography apparatus (Dyax Corp.) on silicagel (32-63 mM, 60 Å pore size) in pre-packed cartridges of the sizenoted. NMR spectra were obtained in CDCl₃ unless otherwise noted.Coupling constants (J) are in hertz (Hz). Abbreviations: diethyl ether(ether), triethylamine (TEA), N,N-diisopropylethylamine (DIEA),tetrahydrofuran (THF), saturated (sat'd), room temperature (rt), hour(s)(h or hr), min(s) (min). For all tables that follow any NMR data followsthe compound.

HPLC Methods

LC-1: Waters Xterra MS C18, 5μ, 4.6×50 mm column, 10:90 to 95:5 v/vCH₃CN/H₂O+0.05% TFA over 4.5 min, hold 1 min, PDA detection 200-600 nm,flow rate=2.5 mL/min.

LC-2: Analytical Sales and Service Armor C8 5μ 20×100 mm column, 10:90to 90:10 v/v CH₃CN/H₂O+0.05% TFA over 12 min, hold 4 min, UV detectionat either 210, 220 or 254 nM, flow rate=10 mL/min.

LC-3: YMC-Pack Pro C18, 5μ, 20 mm×150 mm column, gradient 10:90-80:20v/v CH₃CN:H₂O+0.1% TFA over 23 min then hold at 100:0 v/v CH₃CN:H₂O+0.1%TFA for 7 min; 20 mL/min, 254 nm.

Preparation of Aldehyde Intermediates Aldehyde 1 4-Octyloxybenzaldehyde

4-Hydroxybenzaldehyde (1.00 g, 0.82 mmol), potassium carbonate (1.70 g,12.28 mmol) and 1-iodooctane (2.16 g, 9.00 mmol) were heated together inacetonitrile at 80° C. for 16 h. The reaction was cooled, filtered andconcentrated. Silica gel chromatography eluting with hexane/ethylacetate (20:1) gave a colorless oil (1.63 g): ¹H NMR (500 MHz) δ 9.99(s, 1H), 7.44-7.46 (m, 2H), 7.40 (s, 1H), 7.19 (m, 1H), 4.01 (t, J=6.6Hz, 2H), 1.80 (m, 2H), 1.42-1.50 (m, 2H), 1.24-1.39 (m, 8H), 0.89 (t,J=6.9 Hz, 3H).

Aldehyde 2 4-Hydroxy-3-propyloxybenzaldehyde

3,4-Dihydroxybenzaldehyde (0.5 g, 3.62 mmol) was dissolved in DMF (10mL) and sodium hydride (0.087 g, 3.62 mmol) was added. The reactionmixture was stirred at rt for 10 min. Iodopropane (0.35 mL, 0.62 mmol)was added and the reaction was stirred at 80° C. for 2.5 h. The reactionwas diluted with ethyl acetate and washed with 2N HCl and water. Silicagel chromatography eluting with 35% ethyl acetate/hexane yielded 0.16 gof desired product: ESI-MS 181 (M+H).

Aldehyde 3 6-Hydroxy-2-naphthaldehyde

Aluminum trichloride (1.07 g, 8.06 mmol) was added to a solution of6-methoxy-2-naphthaldehyde (1.0 g, 5.37 mmol) in chlorobenzene (15 mL).The reaction mixture was stirred at 130° C. for 4 h. The reaction wasquenched with water (5 mL) and conc. HCl (2 mL). The reaction mixturewas dissolved in ethyl acetate and washed with water and brine and driedover anhydrous magnesium sulfate. Silica gel chromatography eluting with10% ethyl acetate/hexane yielded 0.35 g of desired product: ESI-MS173.0(M+H).

Aldehydes 4-34

The following Aldehydes (4-34) were prepared using a procedure analogousto that described for Aldehyde 1 substituting A for 1-iodooctane and Bfor 4-hydroxybenzaldehyde.

Aldehyde A B ESI-MS 4

249.3 5

277.1 6

265.4 7

263.1 8

269.0 9

279.1 10

11

262.0 12

13

343.0 14

357.1 15

16

¹H NMR (500 MHz, CD₃OD) δ 9.88 (s, 1H), 7.94 (s, 1H), 7.47 (s, 1H), 4.26(t, J=6.3 Hz, 2H), 4.14 (t, J=6.3 Hz, 2H), 4.02 (t, J=6.3 Hz, 2H), 3.25(t, J=6.8 Hz, 2H), 1.76-1.94 (m, 4H), 1.52-1.62 (m, 2H), 0.88-1.00 (m,3H) 17

18

241.1 19

255.2 20

391.1 21

339.3 22

307.3 23

265.2 24

299.1 25

357.1 26

329.0 27

419.1 28

341.3 29

227.1 30

370.9 31

317.1 32

382.7 33

179.1 34

285.1

Aldehyde 35 3-Methoxy-5-methyl-4-octyloxybenzaldehyde

Aldehyde 20 (0.20 g, 0.51 mmol) and tetramethyl tin (0.2 g, 1.12 mmol)were dissolved in N-methyl pyrrolidinone (1 mL) in a sealed tube.Palladium tetrakis(triphenylphosphine) (0.016 g, 0.014 mmol) and copperiodide (0.01 g, 0.05 mmol) were added to the reaction mixture which washeated at 65° for 16 h. The reaction mixture was diluted with ethylacetate and washed with 2N HCl, brine and was dried over magnesiumsulfate. Silica gel chromatography eluting with 10% ethyl acetate/hexanegave desired product: ESI-MS 279.2 (M+H).

Aldehyde 36 3-Methoxy-5-phenyl-4-octyloxybenzaldehyde

Aldehyde 20 (0.25 g, 0.64 mmol), phenylboronic acid (0.12 g, 0.96 mmol),potassium carbonate (0.27 g, 1.92 mmol),tris(dibenzylideneacetone)dipalladium(0) (0.15 g, 0.016 mmol) and2-(dicyclohexylphosphino)biphenyl (0.022 g, 0.064 mmol) were dissolvedin tetrahydrofuran (1 mL). The reaction mixture was stirred at rt for 3h then at 50° C. for 16 h. The reaction mixture was filtered throughcelite. Silica gel chromatography eluting with 10% ethyl acetate/hexanegave desired product: ESI-MS 341.2 (M+H).

Aldehyde 37 3-Hydroxy-4-octyloxybenzaldehyde

Aldehyde 28 (0.25 g, 0.77 mmol) was dissolved in methylene chloride (4mL) and boron tribromiide dimethylsulfide complex (0.6 g, 1.93 mmol) wasadded dropwise. The reaction mixture was stirred at rt for 1 h. Thereaction was quenched with methanol and concentrated in vacuo. Silicagel chromatography eluting with 10% ethyl acetate/hexane yielded 0.155 gof desired product: ESI-MS 251.2 (M+H).

Aldehyde 38 4-(Nonoylamido)benzaldehyde

4-Aminobenzaldehyde (0.3 g, 2.5 mmol) was dissolved in methylenechloride (8 mL) and nonanoyl chloride (0.5 mL, 2.7 mmol) was addedfollowed by DIEA (1.14 mL, 6.25 mmol). The reaction was stirred at rtfor 3 h. Silica gel chromatography eluting with 25% ethyl acetate/hexaneyielded impure product Further purified by HPLC to give 30.0 mg ofdesired product: ESI-MS 262.0 (M+H).

Aldehyde 39 4-(5-Phenylpentyloxy)benzaldehyde

Diethylazodicarboxylate (0.49 g, 2.8 mmol) in tetrahydrofuran (2 mL) wasadded to a solution of 4-hydroxybenzaldehyde (0.25 g, 2.05 mmol),5-phenyl-1-pentanol (0.34 mL, 2.05 mmol) and triphenylphosphine (0.73 g,2.80 mmol) in tetrahydrofuran (10 mL) at rt. The reaction was stirredfor 2 h. The reaction mixture was concentrated in vacuo. Silica gelchromatography eluting with 20% ethyl acetate/hexane yielded 0.070 g ofdesired product: ¹H NMR (500 MHz, CD₃OD): δ 9.83 (s, 1H), 7.86 (d, J=8.7Hz, 2H), 7.25 (t, 2H), 7.14-7.20 (m, 3H), 7.06 (d, J=8.7 Hz, 2H), 4.09(t, J=6.4 Hz, 2H), 2.65 (t, J=7.7 Hz, 2H), 1.80-1.88 (m, 2H), 1.68-1.75(m, 2H), 1.49-1.57 (m, 2H).

Aldehyde 40 3′-Chloro-4′-octyloxy-4-biphenylbenzaldehyde Step A:1-Bromo-3-chloro-4-octyloxybenzene

1-Bromo-3-chloro-4-hydroxybenzene (0.50 g, 2.41 mmol) was dissolved inacetonitrile (20 mL) and stirred at rt. Potassium carbonate (0.47 g,3.37 mmol) and iodooctane (0.57 mL, 3.13 mmol) were added and thereaction was heated to 80° C. for 4 h. The reaction was diluted withethyl acetate, washed with water and dried over anhydrous magnesiumsulfate. Silica gel chromatography eluting with 1% ethyl acetate/hexaneyielded 0.6 g of product: ESI-MS 317.0 (M+H).

Step B: 3′-Chloro-4′-octyloxy-4-biphenylbenzaldehyde

Palladium acetate (0.005 g, 0.022 mmol) and2-(dicyclohexylphosphino)biphenyl (0.015 g, 0.044 mmol) were added to asolution of (4-formylphenyl)boronic acid (0.25 g, 1.65 mmol),1-bromo-3-chloro-4-octoxybenzene (0.35 g, 1.10 mmol, from Step A), andpotassium fluoride (0.19 g, 3.30 mmol) in 1,4-dioxane (3 mL). Thereaction mixture was heated at 75° C. for 3 h. The reaction was cooled,filtered through celite and concentrated in vacuo. Silica gelchromatography eluting with 1% ethyl acetate/hexane yielded 0.17 g ofdesired product: ¹H NMR (500 MHz, CD₃OD): δ 10.01 (s, 1H), 7.97 (d,J=8.0 Hz, 2H), 7.80 (d, J=8.0 Hz, 2H), 7.74 (s, 1H), 7.61 (d, J=7.7 Hz,1H), 7.16 (d, J=8.7 Hz, 1H) 4.11 (t, J=6.2 Hz, 2H), 1.80-1.89 (m, 2H),1.50-1.60 (m, 2H), 1.28-1.46 (m, 8H), 0.88-0.97 (m, 3H)

Aldehydes 41-60

The following Aldehydes (41-60) were made using procedures analogous tothose described for Aldehyde 40 substituting A for 1-iodooctane and Bfor 1-bromo-3-chloro-4-hydroxybenzene in Step A

Aldehyde A B ESI-MS 41

269.1 42

255.0 43

283.1 44

311.0 45

46

311.3 47

331.1 48

313.2 49

255.1 50

269.2 51

52 N/A

259.0 53 N/A

259.0 54 N/A

267.1 55

297.1 56 N/A

253.2 57 N/A

267.1 58 N/A

59

60

Aldehyde 61 4-(Octyloxymethyl)benzaldehyde Step A:4-(Octyloxymethyl)benzyl alcohol

Sodium hydride (0.17 g, 7.20 mmol) was added to a solution of1,4-benzene dimethanol (1.00 g, 7.20 mmol) in THF at 0° C. The reactionwas stirred for 1 h. 1-iodooctane (1.73 g; 7.20 mmol) was added and thereaction mixture was warmed to rt for 4 h and then heated at 50° C. for2 days. The reaction was cooled and filtered. Silica gel chromatographyeluting with 15% ethyl acetate/hexane gave 0.14 g of product: ¹H NMR(500 MHz) δ 7.34-7.40 (m, 4H), 4.68-4.72 (m, 2H, 4.51 (s, 2H), 3.46-3.50(m, 2H), 1.61-1.68 (m, 2H), 1.24-1.40 (m, 10H), 0.88-0.92 (m, 3H).

Step B: 4-(Octyloxymethyl)benzaldehyde

4-(Octyloxymethyl)benzyl alcohol (0.14 g, 0.56 mmol, from Step A) wasdissolved in methylene chloride (1.5 mL) and the reaction mixture wascooled to 0° C. 4-methylmorpholine N-oxide (0.10 g, 0.84 mmol) andmolecular sieves (4A) (0.25 g) were added. Tetrapropylammoniumperruthenate (0.004 g, 0.011 mmol) was added and the resulting mixturewas stirred for 1 h. The reaction mixture was filtered through celite.Silica gel chromatography eluting with 6% ethyl acetate/hexane gave0.018 g of product: ¹H NMR (500 MHz) δ 10.02 (s, 1H), 7.86-7.90 (m, 2H),7.50-7.55 (m, 2H), 4.58-4.62 (s, 2H), 3.50-3.55 (m, 2H), 1.62-1.70 (m,2H), 1.24-1.35 (m, 2H), 0.87-0.93 (m, 2H).

Aldehyde 62 4-(N-Octylcarboxamido)benzaldehyde

DIEA (0.43 mL, 2.33 mmol) was added to a solution of4-carboxybenzaldehyde (0.23 g, 1.55 mmol), octylamine (0.20 g, 1.55mmol) and PyBoP (0.89 g, 1.71 mmol) in methylene chloride (2.5 mL). Thereaction was stirred at rt for 16 h after which it was concentrated.Silica gel chromatography eluting with 25% ethyl acetate/hexane gave0.30 g of product: ESI-MS 262.1 (M+H).

Aldehydes 63-73

The following Aldehydes (63-73) were made using a procedure analogous tothat described for Aldehyde 62 substituting A for octylamine.

Aldehyde A B ESI-MS 63

318.2 64

253.0 65

66

282.2 67

282.2 68

69

¹H NMR (500 MHz): δ 10.10 (s, 1H), 8.20 (d, J=8.2 Hz, 2H), 7.95 (d,J=8.2 Hz, 2H), 4.35 (t, J=6.8 Hz, 2H), 1.75–1.85 (m, 2H), 1.40–1.50 (m,2H), 1.25–1.40 (m, 6H), 0.89 (t, J=7.0 Hz, 3H).

Aldehyde 70 4-(1-Hydroxynon-1-yl)benzaldehyde

Terephthaldicarboxaldehyde (2.00 g, 14.91 mmol) was dissolved intetrahydrofuran (25 mL) and cooled to 0° C. Octylmagnesium chloride (7.5mL, 2.0M in THF, 15 mmol) was added dropwise. After 15 min, the reactionwas quenched with 2N aqueous hydrochloric acid (50 mL) and diluted withethyl acetate (50 mL). The organic layer was separated, washed withsat'd sodium chloride (50 mL), dried over magnesium sulfate andconcentrated in vacuo. Silica gel chromatography eluting with 9% ethylacetate/hexane gave 0.19 g (0.77 mmol, 5.1%) of product: ¹H NMR (500MHz) δ 10.0 (s, 1H), 7.87 (d, J=8.0 Hz, 2H), 7.52 (d, J=8.3 Hz, 2H),4.75-4.80 (m, 1H), 1.68-1.82 (m, 2H), 1.22-1.45 (m, 12H), 0.91 (t, J=7.0Hz, 3H).

Aldehyde 71 4-(1-Nonoyl)benzaldehyde

Dess-Martin periodinane (0.268 g, 0.632 mmol) was added to a solution ofAldehyde 70 (0.125 g, 0.505 mmol) in methylene chloride (3.0 mL). After1 h, the reaction was filtered and concentrated in vacuo. Silica gelchromatography eluting with 5% ethyl acetate/hexane gave 0.107 g (0.446mmol, 88%) of product: ¹H NMR (500 MHz) δ 10.1 (s, 1H), 8.10 (d, J=8.2Hz, 2H), 7.97 (d, J=8.2 Hz, 2H), 3.00 (t, J=7.3 Hz, 2H), 1.70-1.8 (m,2H), 1.22-1.42 (m, 10H), 0.88 (t, J=7.0 Hz, 3H).

Aldehyde 72 4-(1-Decanoyl)benzaldehyde

Tetrakis(triphenylphosphine)palladium(0) (50 mg) was added to a solutionof 4-formylphenylboronic acid (0.50 g, 3.33 mmol), nonanoyl chloride(1.7 mL, 8.33 mmol) and cesium carbonate (2.70 g, 8.33 mmol) in toluene(40 mL) and heated to 80° C. After stirring overnight, the reaction wasdiluted with ethyl acetate (50 mL) and washed with 2N hydrochloric acid(50 mL), sat'd sodium chloride (50 mL), dried over magnesium sulfate andconcentrated in vacuo. Silica gel chromatography eluting with 6% ethylacetate/hexane gave 0.022 g (0.083 mmol, 3%) of product: ¹H NMR (500MHz) δ 10.1 (s, 1H), 8.09 (d, J=8.2 Hz, 2H), 7.98 (d, J=8.2 Hz, 2H),3.00 (t, J=7.4 Hz, 2H), 1.70-1.80 (m, 2H), 1.22-1.42 (m, 12H), 0.88 (t,J=6.9 Hz, 3H).

Aldehyde 73 3-Methyl-4-decanoyl benzaldehyde Step A:4-Bromo-3-methylbenzyl alcohol

DIBALH (1.0M solution in methylene chloride, 31 mL, 31 mmol) was addeddropwise to a solution of methyl 4-bromo-3-methylbenzoate (3.0 g, 14.0mmol) in methylene chloride (20 mL) at 0° C. After 1 h, the reaction wasquenched with 10% aqueous sodium bisulfite (100 mL). The aqueous layerwas separated and extracted with methylene chloride (50 mL). Thecombined organic layers were combined, dried over magnesium sulfate andconcentrated in vacuo. Silica gel chromatography eluting with 17% ethylacetate/hexane gave 1.90 g (9.50 mmol, 68%) of product: ¹H NMR (500 MHz)δ 7.50 (d, J=8.3 Hz, 1H), 7.24 (s, 1H), 7.04 (d, J=8.0 Hz, 1H), 4.62 (d,J=5.7 Hz, 2H), 2.40 (s, 3H).

Step B: 4-(1-Hydroxydec-1-yl)-3-methylbenzyl alcohol

n-Butyllithium (2.5 M in hexanes, 8.3 mL, 20.7 mmol) was added dropwiseto a solution of 4-bromo-3-methylbenzyl alcohol (1.90 g, 9.44 mmol, fromStep A) in tetrahydrofuran (25 mL) at −78° C. After 1 h, n-decanal (2.95g, 18.89 mmol) was added and the reaction allowed to warm to 0° C. After30 min, the reaction was quenched with water (25 mL) and diluted withethyl acetate (25 mL). The organic layer was washed with sat'd sodiumchloride (30 mL), dried over magnesium sulfate and concentrated invacuo. Silica gel chromatography eluting with 25% ethyl acetate/hexanegave 1.69 g (6.07 mmol, 64%) of product: ¹H NMR (500 MHz): δ 7.45 (d,J=8.0 Hz, 1H), 7.21 (d, J=7.8 Hz, 1H), 7.14 (s, 1H), 4.88-4.94 (m, 1H),4.64 (s, 2H), 2.34 (s, 3H), 1.22-1.80 (m, 16H), 0.87 (t, J=7.0 Hz, 3H).

Step C: 3-Methyl-4-decanoyl benzaldehyde

Dess-Martin periodinane (1.00 g, 2.37 mmol) was added to a solution of4-(1-hydroxydec-1-yl)-3-methylbenzyl alcohol (0.300 g, 1.07 mmol, fromStep B) in methylene chloride (5.0 mL). After 20 min, the reaction wasfiltered and concentrated in vacuo. Silica gel chromatography elutingwith 5% ethyl acetate/hexane gave 0.24 g (0.89 mmol, 83%) of product: ¹HNMR (500 MHz) δ 10.0 (s, 1H), 7.76 (d, J=7.8 Hz, 1H), 7.74 (s, 1H), 7.66(d, J=7.8 Hz, 1H), 2.87 (t, J=7.5 Hz, 2H), 2.51 (s, 3H), 1.66-1.74 (m,2H), 1.22-1.38 (m, 12H), 0.87 (t, J=7.0 Hz, 3H).

Aldehyde 74 3-Methyl-4-(4-(nonyl)benzoyl)benzaldehyde

The title compound was prepared using procedures analogous to those usedto prepare Aldehyde 73 substituting 4-(nonyl)benzaldehyde for n-decanalin Step B: ¹H NMR (500 MHz) δ 10.0 (s, 1H), 7.76 (d, J=7.8 Hz, 1H), 7.74(s, 1H), 7.66 (d, J=7.8 Hz, 1H), 2.88 (t, J=7.5 Hz, 2H), 2.51 (s, 3H),1.66-1.74 (m, 2H), 1.22-1.38 (m, 10H), 0.88 (t, J=7.0 Hz, 3H).

Aldehyde 75 3′-(1-Hydroxyhept-1-yl)-4-biphenylcarboxaldehyde Step A:1-Bromo-3-(1-hydroxyhept-1-yl)benzene

Hexylmagnesium bromide (2.0M in THF, 3.7 mL, 7.4 mmol) was added to asolution of 3-bromobenzaldehyde (1.50 g, 8.11 mmol) in tetrahydrofuran(10 mL) at −78° C. After 10 min, the reaction was quenched by theaddition of 2N hydrochloric acid (30 mL) and the product extracted intoethyl acetate (30 mL). The organic layer was washed with sat'd sodiumchloride (25 mL), dried over magnesium sulfate and concentrated invacuo. Silica gel chromatography eluting with 17% ethyl acetate/hexanegave 1.42 g (5.25 mmol, 65%) of product.

Step B: 3′-(1-Hydroxyhept-1-yl)-4-biphenylcarboxaldehyde

To a solution of 1-bromo-3-(1-hydroxyhept-1-yl)benzene (1.00 g, 3.70mmol, from Step A), 4-formylphenylboronic acid (0.83 g, 5.55 mmol) andpotassium fluoride (0.65 g, 11.10 mmol) in tetrahydrofuran (10 mL) wasadded palladium(II) acetate (0.016 g, 0.071 mmol) and2-(dicyclohexylphosphino)biphenyl (0.052 g, 0.148 mmol). After stirringfor 24 h at rt, the reaction was diluted with ethyl acetate (50 mL),washed with water (50 mL), sat'd sodium chloride (50 mL), dried overmagnesium sulfate and concentrated in vacuo. Silica gel chromatographyeluting with 25% ethyl acetate/hexanes gave 0.81 g of product as ayellow oil.

Aldehyde 76 3 ′-(Heptanoyl)-4-biphenylcarboxaldehyde Step A:1-Bromo-3-heptanoyl benzene

Dess-Martin periodinane (4.40 g, 15% solution in methylene chloride,1.56 mmol) was added to a solution of1-bromo-3-(1-hydroxyhept-1-yl)benzene (0.39 g, 1.42 mmol, from Aldehyde75, Step A). After 1 h, the reaction was quenched by the addition of 1Nsodium hydroxide (20 mL). The aqueous layer was separated, washed withmethylene chloride (20 mL) and the organic layers combined, dried overmagnesium sulfate and concentrated in vacuo. Silica gel chromatographyeluting with 5% ethyl acetate/hexane gave 0.30 g (1.11 mmol, 78%) ofproduct: ¹H NMR (500 MHz) δ 8.08 (t, J=1.7 Hz, 1H), 7.87 (d, J=7.7 Hz,1H), 7.68 (d, J=8.0 Hz, 1H), 7.34 (t, J=7.9 Hz, 1H), 2.93 (t, J=7.4 Hz,2H), 1.68-1.76 (m, 2H), 1.28-1.40 (m, 6H), 0.89 (t, J=7.0 Hz, 3H).

Step B: 3′-(Heptanoyl)-4-biphenylcarboxaldehyde

To a solution of 1-bromo-3-heptanoyl benzene (0.30 g, 1.11 mmol, fromStep A), 4-formylphenylboronic acid (0.25 g, 1.68 mmol) and potassiumfluoride (0.20 g, 3.36 mmol) in tetrahydrofuran (2.5 mL) was addedpalladium(II) acetate (0.006 g, 0.025 mmol) and2-(dicyclohexylphosphino)biphenyl (0.016 g, 0.050 mmol). After stirringfor 3 h at 50° C., the reaction was placed onto silica gel and elutedwith 10% ethyl acetate/hexanes to give 0.26 g (0.88 mmol, 80%) ofproduct as a yellow oil: ¹H NMR (500 MHz) δ 8.22 (t, J=1.7 Hz, 1H),7.90-8.10 (m, 3H), 8.30 (d, J=8.0 Hz, 1H), 7.99 (d, J=8.3 Hz, 2H), 7.58(t, J=7.8 Hz, 1H), 3.02 (t, J=7.4 Hz, 2H), 1.66-1.80 (m, 2H), 1.38-1.44(m, 2H), 1.30-1.38 (m, 4H), 0.90 (t, J=7.0 Hz, 3H).

Aldehyde 77 3-(Cyclopropyloxy)-4-(nonyloxy)benzaldehyde

To a solution of 1.78 g (10.0 mmol) of3-(cyclopropyloxy)-4-hydroxybenzaldehyde and 2.54 g (10.0 mmol) of1-iodononane in 20 mL acetonitrile was added 3.58 g (11.0 mmol) ofCs₂CO₃. The slurry was stirred at rt for 12 h. The reaction was quenchedwith 30 mL of water and extracted with ethyl acetate (50 mL×2). Thecombined extractions were washed with water, dried with sodium sulfateand concentrated to a solid. Flash chromatography on a Biotage 40Mcartridge using 10% ethyl acetate/hexanes afforded 2.9 g (95%) of thetitle compound as a white solid. ¹H NMR (500 Mhz) δ 0.87-0.91 (m, 7H),1.30-1.90 (m, 14H), 3.85 (m, 1H), 4.10 (t, J=6.9, 2H), 6.98 (d, J=8.2,1H), 7.48 (dd, J=8.5, 1.8, 1H), 7.77 (d, J=1.8, 1H), 9.89 (s, 1H); LC-1:4.6 min; ESI-MS 305 (M+H).

Aldehyde 78 4-(Nonylthio)benzaldehyde

To a solution of 3.15 g (10.0 mmol) of 1-bromo-4-(nonylthio)benzene in50 mL anhydrous THF was slowly added 9.4 mL of n-BuLi (1.6 M in hexanes,15 mmol) at −50° C. The mixture was aged at the same temperature for 1 hbefore the addition of 2.3 mL of anhydrous DMF. The reaction mixture wasallowed to warm to 0° C. and was quenched with 2 N HCl to pH=1. Thelayers were separated and the aqueous layer was extracted with ethylacetate (50 mL×2). The combined organic layer and extractions werewashed with water and concentrated to oil. Flash chromatography on aBiotage 40M cartridge using 5% ethyl acetate/hexanes afforded 2.35 g(89%) of the title compound as light yellow oil: ¹H NMR (500 MHz) δ 0.91(t, J=7.0, 3H), 1.30-1.76 (m, 14H), 3.03 (t, J=7.4, 2H), 7.37 (d, J=8.5,2H), 7.78 (d, J=8.5, 2H), 9.95 (s, 1H); LC-1: 4.8 min; ESI-MS 265 (M+H).

Aldehyde 793-(4-(Formyl)phenyl)-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazoleStep A: (E/Z)-2-Phenyl-3-chloro-4,4,4-trifluoro-2-butanal

Phosphorous oxychloride (7.5 mL, 80 mmol) was added to 15 mL of DMF at0° C. The resulting mixture was warmed to rt and stirred for 1 h. Asolution of 5.0 g (26.6 mmol) of1,1,1-trifluoromethyl-3-phenyl-2-propanone in 1 mL of DMF was added andthe resulting mixture was stirred at 70° C. for 20 h. The reactionmixture was cooled to rt, poured onto 150 g of ice and stirred atambient temperature for 1 h. The quenched mixture was extracted with 200mL of ether. The extract was washed with 200 mL of water, dried andconcentrated. Chromatography on a Biotage 40 M cartridge using hexanes(4 L) as the eluant afforded 5:1 g (82%) of the title compound.

Step B: Ethyl(4-phenyl-5-trifluoromethyl)thiophene-2-carboxylate

Ethyl mercaptoacetate (2.75 mL, 25.0 mmol) was added to a suspension of600 mg (25 mmol) of NaH in 45 mL of THF maintaining the internaltemperature at 25° C. A solution of 5.10 g (21.7 mmol) of(E/Z)-2-phenyl-3-chloro-4,4,4-trifluoro-2-butanal (from Step A) wasadded and the resulting mixture was stirred at rt for 20 h. The reactionwas quenched with 50 mL of sat'd NH₄Cl and the resulting mixture waspartitioned between 250 mL of ether and 100 mL of water. The organiclayer was separated, dried and concentrated. Chromatography on a Biotage40 M cartridge using hexanes (1 L), then 4:1 v/v hexanes/CH₂Cl₂ (1 L) asthe eluant afforded 5.10 g (78%) of the title compound: ¹H NMR (400 Mhz)δ 1.40 (t, J=7.2, 3H), 4.39 (q, J=7.2, 2H), 7.42 (app s, 5H), 7.74 (q,J=1.6, 1H).

Step C: (4-Phenyl-5-trifluoromethyl)thiophene-2-carboxylic acid

A solution of 5.10 g (17.0 mmol) of ethyl4-phenyl-5-trifluoromethyl-thiophene-2-carboxylate (from Step B) in 20mL of EtOH was treated with 10 mL of 5.0 N NaOH and stirred at rt for 30min. The EtOH was removed in vacuo. The residual aqueous mixture wasacidified to pH 2 with 1 N HCl, then extracted with 300 mL of 1:1 v/vEtOAc/ether. The extract was separated, dried and concentrated.Recrystallization from 200 mL of 20:1 v/v hexanes/ether afforded 4.30 g(93%) of the title compound: ¹H NMR (500 MHz) δ 7.43 (app s, 5H), 7.84(app s, 1H); ¹³C NMR (CDCl₃, 125 MHz) δ 121.7 (q, J=269), 128.5, 128.6,128.8, 132.5 (q, J=36), 133.3, 133.8, 137.5, 144.8, 167.0.

Step D:3-[4-(Carbomethoxy)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole

A solution of 408 mg (1.5 mmol) of4-phenyl-5-trifluoromethyl-thiophene-2-carboxylic acid and 1 mL ofoxalyl chloride in 5 mL of CH₂Cl₂ was treated with 5 drops of DMF. Theresulting mixture was stirred at rt for 1 h, then concentrated. Thecrude acid chloride and 291 mg (1.5 mmol) of4-(carbomethoxy)benzaridoxime were dissolved in 7 mL of 6:1 v/vxylenes/pyridine. The resulting solution was heated at 140° C. for 1 h,then cooled. The mixture was partitioned between 50 mL of 1:1EtOAc/ether and 50 mL of 1 N HCl. The organic layer was separated,washed with 3×50 mL of 1 N HCl, 50 mL of sat'd NaHCO₃, dried andconcentrated. Chromatography on a Biotage 40 M cartridge using hexanes(1 L), then 20:1 v/v hexanes/EtOAc (1 L) as the eluant afforded 423 mg(65%) of the title compound: ¹H NMR (500 MHz) δ 3.97 (s, 3H), 7.48 (apps, 5H), 7.92 (s, 1H), 8.18 (app d, J=8.5, 2H), 8.23 (app d, J=8.5, 2H).

Step E:3-[4-(Hydroxymethyl)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole

A solution of 390 mg (0.91 mmol) of3-[4-(carbomethoxy)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole(from Step D) in 10 mL of CH₂Cl₂ at −78° C. was treated with 2.7 mL of1.0 M DIBALH solution in CH₂Cl₂. The resulting solution was stirred coldfor 1 h, then quenched with 5 mL of sat'd Rochelle salt solution. Themixture was partitioned between 100 mL CH₂Cl₂ and 50 mL of 1 N NaOH. Theorganic layer was separated, dried and concentrated. Chromatography on aBiotage 40 S cartridge using 4:1 v/v hexanes/EtOAc (1 L) as the eluantafforded 325 mg (89%) of the title compound: ¹H NMR (500 Mhz) δ 1.80(app s, 1H), 4.80 (d, J=4.0, 2H), 7.46-7.48 (5H), 7.52 (d, J=8.0, 2H),7.91 (q, J=1.5, 1H), 8.14 (d, J=8.0, 2H).

Step F:3-[4-(Formyl)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole

A mixture of 310 mg (0.77 mmol) of3-[4-(hydroxymethyl)phenyl]-5-(4-phenyl-5-trifluoromethyl-2-thienyl)-1,2,4-oxadiazole(from Step E), 527 mg (1.5 mmol) of 4-methylmorpholine N-oxide and 500mg of 4 A molecular sieves in 15 mL of CH₃CN was treated with 12 mg(0.034 mmol) of tetrapropylammonium perruthenate and the resultingmixture was stirred ar rt for 2 h. The solids were filtered and thefiltrated was concentrated. Chromatography on a Biotage 40 S cartridgeusing 9:1 v/v hexanes/EtOAc (1 L) as the eluant afforded 205 mg (66%) ofthe title compound: ¹H NMR (500 MHz) δ 7.48 (app s, 5H), 7.93 (app s,1H), 8.03 (d, J=8.5, 2H), 8.33 (d, J=8.5, 2H), 10.1 (s, 1H).

Aldehyde 804-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzaldehyde Step A:2-Hydroxymethylphenyl-5-trifluoromethyl-thiophene

A solution of 2.10 g (7.7 mmol) of4-phenyl-5-trifluoromethyl-thiophene-2-carboxylic acid (from Aldehyde17, Step C) in 20 mL of THF was treated with 5.0 mL of 2.0 M boranedimethylsulfide complex in THF. The resulting solution was heated atreflux for 3 h, cooled to rt, quenched with 10 mL of MeOH andconcentrated. Chromatography on a Biotage 40M cartridge using 9:1 v/vhexanes/EtOAc as the eluant afforded 1.95 g (98%) of the title compound:¹H NMR (500 MHz) δ 2.05 (app s, 1H), 4.87 (s, 2H), 6.99 (s, 1H), 7.41(app s, 5H).

Step B: 4-((4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzaldehyde

A solution of 1.95 g (7.5 mmol) of2-hydroxymethyl-4-phenyl-5-trifluoromethyl-thiophene (from Step A), 925mg (7.6 mmol) of 4-hydroxybenzaldehyde and 3.0 g (11.4 mmol) oftriphenylphosphene in 40 mL of THF at 0° C. was treated with 2.0 g (11.4mmol) of diethylazodicarboxylate. The resulting mixture was warmed tort, stirred for 2 h, then concentrated. Chromatography on a Biotage 75Scartridge using 9:1 v/v heptane/EtOAc as the eluant afforded 2.5 g ofimpure title compound. Chromatography on a Biotage 40M cartridge using19:1 v/v hexanes/EtOAc (1 L), then 4:1 v/v hexanes/EtOAc (1 L) as theeluant afforded 1.65 g (60%) of the title compound: ¹H NMR (500 Mhz) δ5.32 (s, 2H), 7.10 (d, J=8.5, 2H), 7.12 (s, 1H), 7.41-7.43 (5H),7.85-7.90 (2H), 9.92 (s, 1H).

PREPARATION OF EXAMPLES Example 1N-((4-Decyloxy)benzyl)-3-aminopropylphosphonic acid

3-Aminopropylphosphonic acid (0.064 g, 0.457 mmol) andtetrabutylammonium hydroxide (1.0M in methanol, 0.46 mL, 0.46 mmol) inmethanol (3 mL) were heated at 50° C. for 1 h to dissolve all solids.4-(Decyloxy)benzaldehyde (0.100 g, 0.381 mmol) and sodiumcyanoborohydride (0.025 g, 0.40 mmol) were added and stirring wascontinued for 1 h at 50° C. The reaction mixture was made acidic (pH˜5)by the addition of concentrated HCl then directly purified by LC-3 togive the title compound (0.055 g): ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d,J=8.7 Hz, 2H), 6.98 (d, J=8.7 Hz, 2H), 4.12 (s, 2H), 3.99 (t, J=6.4 Hz,2H), 3.12 (t, J=7.7 Hz, 2H), 2.0 (m, 2H), 1.64-1.84 (m, 4H), 1.47 (m,2H), 1.24-1.40 (m, 12H), 0.90 (t, J=6.9 Hz, 3H); MS m/e 386.4 (M+H).

Examples 2-107

The following Examples (2-112) were prepared using a procedure analogousto that described in EXAMPLE 1 substituting A for4-(decyloxy)benzaldehyde and B for 3-aminopropylphosphonic acid.

EXAMPLE A B ESI-MS 2

358.2 ¹H NMR (500 MHz, CD₃OD) δ 7.35-7.41 (m, 2H), 6.94-7.01 (m, 2H),4.08-4.13 (m, 2H), 3.96–4.02 (m, 2H), 3.08-3.14 (m, 2H), 1.93-2.04 (m,2H), 1.73-1.82 (m, 4H), 1.43-1.51 (m, 2H), 1.26-1.41 (m, 8H), 0.87-0.94(m, 3H). 3

372.2 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, 2H), 6.98 (d, 2H), 4.86 (s,19H), 4.12 (s, 2H), 3.98 (t, 2H), 3.12 (t, 2H), 1.94-2.04 (m, 2H),1.72-1.84 (m, 4H), 1.42-1.52 (m, 2H), 1.24-1.41 (m, 8H), 0.90 (t, 3H). 4

400.2 ¹H NMR (500 MHz, CD₃OD) δ 7.36-7.40 (m, 2H), 6.95-7.01 (m, 2H),4.12 (s, 2H), 3.95-4.02 (m, 2H), 3.09-3.15 (m, 2H), 1.94-2.04 (m, 2H),1.72-1.84 (m, 4H), 1.42-1.52 (m, 2H), 1.24-1.42 (m, 8H), 0.87-0.94 (m,3H). 5

336.2 ¹H NMR (500 MHz, CD₃OD) δ 7.33-7.44 (m, 5H), 7.27-7.33 (m, 2H),7.03-7.09 (m, 2H), 5.11 (s, 2H), 4.11 (s, 2H), 3.07-3.15 (m, 2H),1.92-2.04 (m, 2H), 1.73-1.82 (m, 2H). 6

372.2 ¹H NMR (500 MHz, CD₃OD) δ 7.42-7.50 (m, 4H), 4.52 (s, 2H), 4.18(s, 2H), 3.46- 3.52 (m, 2H), 3.11-3.18 (m, 2H), 1.95-2.06 (m, 2H),1.75-1.85 (m, 2H), 1.56-1.64 (m, 2H), 1.25-1.34 (m, 6H), 0.85-0.92 (m,3H). 7

358.2 ¹H NMR (500 MHz, CD₃OD) δ 7.34 (t, J=7.9 Hz, 1H), 7.05 (d, J=2.3Hz, 1H), 7.03 (d, J=7.8 Hz, 1H), 6.98 (dd, J=2.3, 8.4 Hz), 4.12 (s, 2H),4.00 (t, J=6.5 Hz, 2H), 3.12 (t, J=6.9 Hz, 2H), 1.94-2.20 (m, 2H),1.70-1.82 (m, 4H), 1.44-1.52 (m, 2H), 1.26-1.40 (m, 8H), 0.90 (t, J=6.9Hz, 3H). 8

342.3 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.0 Hz, 2H), 7.29 (d, J=8.0Hz, 2H), 4.15 (s, 2H), 3.14 (t, J=7.7 Hz, 2H), 2.64 (t, J=7.7 Hz, 2H),2.00 (m, 2H), 1.81 (td, J=7.6, 18.5 Hz, 2H), 1.58-1.64 (m, 2H),1.22-1.36 (m, 10H), 0.89 (t, J=7.0 Hz, 3H). 9

370.1 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.15 (s, 2H), 3.14 (t, J=7.7 Hz, 2H), 2.64 (t, J=7.6 Hz, 2H),2.00 (m, 2H), 1.80 (td, J=7.6, 18.5 Hz, 2H), 1.56-1.64 (m, 2H),1.24-1.38 (m, 14H), 0.89 (t, J=7.0 Hz, 3H). 11

306.1 ¹H NMR (500 MHz, CD₃OD) δ 7.72 (m, 2H), 7.63 (m, 2H), 7.56 (m,2H), 7.45 (m, 2H), 7.36 (m, 1H), 4.24 (s, 2H), 3.18 (t, 2H), 1.97-2.08(m, 2H), 1.76-1.86 (m, 2H). 12

354.2 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, J=8.3 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.15 (s, 2H), 3.12 (t, J=7.3 Hz, 2H), 2.64 (t, J=7.6 Hz, 2H),1.98 (m, 2H), 1.76-1.84 (m, 2H), 1.58-1.64 (m, 2H), 1.43 (d, J=14 Hz,3H), 1.24-1.36 (m, 12H), 0.89 (t, J=7.0 Hz, 3H). 13

400.1 ¹H NMR (500 MHz, CD₃OD) δ 7.41 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.14- 4.22 (m, 2H), 4.04 (t, J=6.0 Hz, 1H), 2.64 (t, J=7.6 Hz,2H), 2.20-2.30 (m, 2H), 1.74- 1.98 (m, 2H). 1.58-1.64 (m, 2H), 1.24-1.32(m, 12H), 0.90 (t, J=7.0 Hz, 3H). 14

370.3 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.1Hz, 2H), 4.15 (s, 2H), 3.05 (t, J=7.8 Hz, 2H), 2.64 (t, J=7.7 Hz, 2H),1.58-1.984 (m, 8H), 1.24-1.36 (m, 12H), 0.89 (t, J=7.0 Hz, 3H). 15

320.2 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, J=8.0 Hz, 2H), 7.29 (d, J=8.0Hz, 2H), 4.15 (s, 2H), 3.10 (t, J=7.8 Hz, 2H), 2.64 (t, J=7.7 Hz, 2H),2.45 (t, J=7.0 Hz, 2H), 1.93- 1.99 (m, 2H). 1.56-1.64 (m, 2H), 1.24-1.34(m, 12H), 0.89 (t, J=7.0 Hz, 3H). 16

336.2 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, J=8.1 Hz, 2H), 7.28 (d, J=8.3Hz, 2H), 4.26 (dd, J=4.1, 7.8 Hz, 1H), 4.17 (s, 2H), 3.16-3.22 (m, 2H),2.64 (t, J=7.7 Hz, 2H), 2.16- 2.24 (m, 1H), 1.98-2.06 (m, 1H), 1.58-1.64(m, 2H), 1.24-1.32 (m, 12H), 0.89 (t, J=7.0 Hz, 3H). 17

336.2 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.26 (dd, J=4.1, 8.0 Hz, 1H), 4.17 (s, 2H), 3.16-3.22 (m, 2H),2.64 (t, J=7.7 Hz, 2H), 2.16- 2.24 (m, 1H), 1.98-2.06 (m, 1H), 1.58-1.64(m, 2H), 1.24-1.32 (m, 12H), 0.89 (t, J=7.0 Hz, 3H) 18

350.2 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.26 (dd, J=4.3, 8.0 Hz, 1H), 4.17 (s, 2H), 3.16-3.22 (m, 2H),2.64 (t, J=7.7 Hz, 2H), 2.16- 2.24 (m, 1H), 1.98-2.06 (m, 1H), 1.58-1.64(m, 2H), 1.24-1.32 (m, 14H), 0.89 (t, J=7.0 Hz, 3H) 19

344.2 ¹H NMR (500 MHz, CD₃OD) δ 7.38 (d, J=7.0 Hz, 2H), 7.28 (d, J=7.8Hz, 2H), 4.18 (s, 2H), 3.17 (t, J=7.4 Hz, 2H), 3.06 (t, J=7.4 Hz, 2H),2.64 (t, J=7.6 Hz, 2H), 2.20 (m, 2H), 1.56-1.64 (m, 2H), 1.22-1.36 (m,12H), 0.89 (t, J=7.0 Hz, 3H) 20

356.2 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.0 Hz, 2H), 7.29 (d, J=8.2Hz, 2H), 7.03 (d, J=7.8 Hz, 1H), 4.20 (s, 2H), 2.65 (t, J=7.7 Hz, 2H),2.49-2.60 (m, 2H), 1.58-1.64 (m, 2H), 1.24-1.34 (m, 14H), 0.89 (t, J=7.0Hz, 3H) 21

336.3 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.3Hz, 2H), 4.25- 4.31 (m, 1H), 4.19 (s, 2H), 3.18 dd, J=2.9, 12.5 Hz, 1H),2.98 (dd, J=9.9, 12.6 Hz, 1H), 2.64 (t, J=7.7 Hz, 2H), 2.53 (d, J=6.2Hz, 2H), 1.56-1.64 (m, 2H), 1.24-1.34 (m, 12H), 0.89 (t, J=7.0 Hz, 3H)22

338.2 ¹H NMR (500 MHz, CD₃OD) δ 7.40 (d, J=8.0 Hz, 2H), 7.30 (d, J=7.7Hz, 2H), 5.14- 5.32 (m, 1H), 4.23 (m, 2H), 3.34-3.42 (m, 2H), 2.74-2.82(m, 2H), 2.65 (t, J=7.7 Hz, 2H), 1.56-1.63 (m, 2H), 1.24-1.36 (m, 12H),0.89 (t, J=7.0 Hz, 3H) 23

388.1 ¹H NMR (500 MHz, CD₃OD) δ 7.24-7.28 (m, 1H), 6.60-6.63 (m, 1H),6.53-6.57 (m, 1H), 4.11 (s, 2H), 3.96-4.02 (m, 2H), 3.88-3.92 (m, 3H),3.28-3.33 (m, 2H), 3.06-3.12 (m, 2H), 1.94-2.05 (m, 2H), 1.72-1.82 (m,4H), 1.43-1.52 (m, 2H), 1.26-1.41 (m, 8H), 0.87-0.94 (m, 3H) 24

386.2 ¹H NMR (500 MHz, CD₃OD) δ 6.68 (s, 2H), 4.20-4.25 (m, 2H),3.91-3.97 (m, 2H), 3.22-3.27 (m, 2H), 2.41 (s, 6H), 1.99-2.10 (m, 2H),1.78-1.87 (m, 2H), 1.69-1.78 (m, 2H), 1.41-1.50 (m, 2H), 1.26-1.40 (m,8H), 0.86-0.94 (m, 3H) 25

516.1 ¹H NMR (500 MHz, CD₃OD) δ 7.78 (s, 2H), 4.14 (s, 2H), 4.00–4.05(m, 2H), 3.12- 3.18 (m, 2H), 1.94-2.04 (m, 2H), 1.76-1.90 (m, 4H),1.52-1.59 (m, 2H), 1.29-1.44 (m, 8H), 0.88-0.94 (m, 3H) 26

392.2 ¹H NMR (500 MHz, CD₃OD) δ 7.52-7.54 (m, 1H), 7.34-7.38 (m, 1H),7.08-7.13 (m, 1H), 4.04-4.14 (m, 4H), 3.09-3.16 (m, 2H), 1.93-2.04 (m,2H), 1.73-1.85 (m, 4H), 1.46-1.55 (m, 2H), 1.26-1.42 (m, 8H), 0.87-0.94(m, 3H) 27

408.3 ¹H NMR (500 MHz, CD₃OD) δ 8.35-8.38 (m, 1H), 8.05-8.09 (m, 1H),7.64-7.70 (m, 1H), 7.54-7.62 (m, 2H), 6.94-6.98 (m, 1H), 4.61 (s, 2H),4.18–4.24 (m, 2H), 3.21-3.27 (m, 2H), 1.99-2.08 (m, 2H), 1.91-1.99 (m,2H), 1.75-1.85 (m, 2H), 1.55-1.64 (m, 2H), 1.27-1.48 (m, 8H), 0.87-0.94(m, 3H) 28

402.2 ¹H NMR (500 MHz, CD₃OD) δ 7.05-7.08 (m, 1H), 6.98-7.01 (m, 2H),4.06–4.14 (m, 3H), 3.98-4.04 (m, 2H), 3.28-3.32 (m, 2H), 3.08-3.15 (m,2H), 1.94-2.04 (m, 2H), 1.72-1.84 (m, 4H), 1.45-1.52 (m, 2H), 1.38-1.44(m, 2H), 1.26-1.38 (m, 8H), 0.86-0.94 29

372.3 ¹H NMR (500 MHz, CD₃OD) δ 7.22-7.27 (m, 2H), 6.91-6.95 (m, 1H),4.07 (s, 2H), 3.97–4.03 (m, 2H), 3.07-3.14 (m, 2H), 2.22 (s, 3H),1.93-2.04 (m, 2H), 1.73-1.84 (m, 4H), 1.46-1.54 (m, 2H), 1.26-1.42 (m,8H), 0.86-0.93 (m, 3H) 30

¹H NMR (500 MHz, CD₃OD) δ 7.19-7.28 (m, 2H), 7.11-7.16 (m, 1H), 4.11 (s,2H), 4.03–4.08 (m, 2H), 3.09-3.15 (m, 2H), 1.93-2.04 (m, 2H), 1.72-1.84(m, 4H), 1.44-1.54 (m, 2H), 1.26-1.42 (m, 8H), 0.86-0.94 (m, 3H) 31

392.1 ¹H NMR (500 MHz, CD₃OD) δ 7.48 (d, J=8.5 Hz, 1H), 7.09 (d, J=2.3Hz, 1H), 6.96 (dd, J=2.6, 8.6, 1H), 4.28 (s, 2H), 4.00 (t, J=6.4 Hz,2H), 3.29-3.30 (m, 2H), 3.18 (t, J=7.4 Hz, 2H0, 1.97-2.08 (m, 2H),1.73-1.84 (m, 4H0, 1.42-1.52 (m, 2H), 1.26-1.41 (m, 8H), 0.87-0.94 (m,3H) 32

385.4 ¹H NMR (500 MHz, CD₃OD) δ 7.86-7.91 (m, 2H), 7.56-7.60 (m, 2H),4.24 (s, 2H), 3.34-3.40 (m, 2H), 3.14-3.19 (m, 2H), 1.95-2.07 (m, 2H),1.74-1.84 (m, 2H), 1.58-1.67 (m, 2H), 1.25-1.43 (m, 10H), 0.86-0.92 (m,3H) 33

441.5 ¹H NMR (500 MHz, CD₃OD) δ 7.56-7.60 (m, 2H), 7.42-7.46 (m, 2H),4.23 (s, 2H), 3.46-3.52 (m, 2H), 3.20-3.26 (m, 2H), 3.14-3.20 (m, 2H),1.94-2.06 (m, 2H), 1.73-1.84 (m, 2H), 1.64-1.72 (m, 2H), 1.45-1.56 (m,2H), 1.32-1.44 (m, 8H), 1.18-1.27 (m, 2H), 1.04-1.18 (m, 2H), 0.88-0.98(m, 3H), 0.80-0.88 (m, 3H) 34

391.2 ¹H NMR (500 MHz, CD₃OD) δ 7.85 (d, J=8.3 Hz, 2H), 7.57 (d, J=8.2Hz, 2H), 7.12 (d, J=8.1 Hz, 2H), 7.09 (d, J=8.0 Hz, 2H), 4.25 (s, 2H),3.58 (t, J=7.4 Hz, 2H), 3.17 (t, J=7.6 Hz, 2H), 2.87 (t, J=7.5, 2H),2.28 (s, 3H), 1.98-2.03 (m, 2H), 1.79-1.84 (m, 2H) 35

431.1 ¹H NMR (500 MHz, CD₃OD) δ 7.95 (d, J=8.3 Hz, 2H), 7.63 (d, J=8.0,2H), 7.60 (d, J=8.2, 2H), 7.54 (d, J=8.0 Hz, 2H), 4.65 (s, 2H), 4.26 (s,2H). 3.17 (t, J=7.3, 2H), 1.98- 2.06 (m, 2H), 1.75-1.84 (m, 2H) 36

459.2 37

405.2 ¹H NMR (500 MHz, CD₃OD) δ 7.88 (d, J=8.2 Hz, 2H), 7.57 (d, J=8.2Hz, 2H), 7.23 (t, J=7.5, 2H), 7.18 (d, J=7.1, 2H), 7.13 (t, J=7.2 Hz,1H), 4.24 (s, 2H), 3.37-3.43 (m, 2H), 3.13-3.20 (m, 2H), 2.62-2.70 (m,2H), 1.95-2.06 (m, 2H), 1.74-1.84 (m, 2H), 1.60-1.74 (m, 4H) 38

334.2 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.2 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.21 (d, J=13.0 Hz, 1H), 4.18 (d, J=13.0 Hz, 1H), 3.32-3.40 (m,1H), 2.64 (t, J=7.7 Hz, 2H), 2.52 (ddd, J=16.9, 7.5, 6.2 Hz, 1H), 2.43(dt, J=17.2, 7.7 Hz, 1H), 2.12-2.20 (m, 1H), 1.76-1.86 (m, 1H),1.56-1.65 (m, 2H), 1.38 (d, J=6.7 Hz, 3H), 1.22-1.34 (m, 12H), 0.90 (t,J=6.3 Hz, 3H). 39

370.2 ¹H NMR (500 MHz, CD₃OD) δ 7.37 (d, J=8.2 Hz, 2H), 7.30 (d, J=8.2Hz, 2H), 4.33 (q, J=6.8 Hz, 1H), 3.00-3.08 (m, 1H), 2.82-2.88 (m, 1H),2.64 (t, J=7.7 Hz, 2H), 1.90- 2.00 (m, 2H), 1.70-1.80 (m, 2H), 1.65 (d,J=6.9 Hz, 3H), 1.58-1.64 (m, 2H), 1.22-1.36 (m, 12H), 0.89 (t, J=6.9 Hz,3H). 40

350.1 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.24- 4.30 (m, 1H), 4.19 (s, 2H), 3.17 (dd, J=12.6, 3.0 Hz,1H), 2.98 (dd, J=12.9, 9.9 Hz, 1H), 2.64 (t, J=7.7 Hz, 2H), 2.52 (d,J=6.1 Hz, 2H), 1.58-1.65 (m, 2H), 1.24-1.35 (m, 14H), 0.89 (t, J=7.0 Hz,3H). 41

336.2 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.24- 4.30 (m, 1H), 4.19 (s, 2H), 3.17 (dd, J=12.6, 3.1 Hz,1H), 2.98 (dd, J=12.9, 9.8 Hz, 1H), 2.64 (t, J=7.7 Hz, 2H), 2.52 (d,J=6.1 Hz, 2H), 1.58-1.65 (m, 2H), 1.24-1.35 (m, 12H), 0.89 (t, J=6.9 Hz,3H). 42

366.2 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.7 Hz, 2H), 6.98 (d, J=8.7Hz, 2H), 4.25- 4.30 (m, 1H), 4.16 (s, 2H), 3.99 (t, J=6.5 Hz, 2H), 3.16(dd, J=12.5, 2.9 Hz, 1H), 2.96 (dd, J=12.8, 9.8 Hz, 1H), 2.52 (d, J=6.2Hz, 2H), 1.74-1.80 (m, 2H), 1.44-1.51 (m, 2H), 1.22-1.40 (m, 12H), 0.90(t, J=7.0 Hz, 3H). 43

388.1 ¹H NMR (500 MHz, CD₃OD) δ 8.35 (d, J=8.5 Hz, 1H), 8.09 (d, J=8.5Hz, 1H), 7.67 (t, J=8.4 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.57 (t, J=8.0Hz, 1H), 6.96 (d, J=8.0 Hz, 1H), 4.66 (s, 2H), 4.32-4.38 (m, 1H) 4.21(t, J=6.4 Hz, 2H), 3.26-3.32 (m, 1H), 3.08 (dd, J=12.8, 9.8 Hz, 1H),2.55 (d, J=6.2 Hz, 2H), 1.91-1.98 (m, 2H), 1.56-1.62 (m, 2H), 1.28-1.48(m, 8H), 0.90 (t, J=6.9 Hz, 3H). 44

366.2 ¹H NMR (500 MHz, CD₃OD) δ 6.69 (s, 2H), 4.35-4.40 (m, 1H), 4.33(d, J=13.8 Hz, 1H), 4.26 (d, J=13.7 Hz, 1H), 3.95 (t, J=6.5 Hz, 2H),3.30-3.35 (m, 1H), 3.09 (dd, J=12.8, 9.9 Hz, 1H), 2.56 (d, J=6.2 Hz,2H), 2.42 (s, 6H), 1.71-1.78 (m, 2H), 1.42-1.48 (m, 2H), 1.28-1.38 (m,8H), 0.90 (t, J=7.0 Hz, 3H). 45

372.2 ¹H NMR (500 MHz, CD₃OD) δ 8.12 (d, J=8.3 Hz, 2H), 7.65 (d, J=8.2Hz, 2H), 4.36 (t, J=6.6 Hz, 2H), 4.30 (s, 2H), 3.21 (t, J=7.5 Hz, 2H),2.00-2.10 (m, 4H), 1.32-1.52 (m, 8H), 0.93 (t, J=7.0 Hz, 3H). 46

372.2 47

370.2 ¹H NMR (500 MHz, CD₃OD) δ 8.06 (d, J=8.3 Hz, 2H), 7.65 (d, J=8.3Hz, 2H), 4.23 (s, 2H), 3.16 (t, J=6.1 Hz, 2H), 3.04 (t, J=7.4 Hz, 2H),1.96-2.06 (m, 2H), 1.66-1.78 (m, 4H), 1.26-1.44 (m, 10H), 0.91 (t, J=7.1Hz, 3H). 48

368.3 ¹H NMR (500 MHz, CD₃OD) δ 7.41 (d, J=8.0 Hz, 2H), 7.30 (d, J=8.0Hz, 2H), 4.18 (s, 2H), 3.16 (t, J=7.4 Hz, 2H), 2.67 (t, J=7.7 Hz, 2H),1.96-2.06 (m, 2H), 1.82-1.88 (m, 2H), 1.60-1.68 (m, 2H), 1.59 (d, J=14.2Hz, 3H), 1.26-1.36 (m, 14H), 0.92 (t, J= 7.0 Hz, 3H). 49

334.2 ¹H NMR (500 MHz, CD₃OD) δ 7.40 (d, J=8.1 Hz, 2H), 7.31 (d, J=8.0Hz, 2H), 4.18 (s, 2H), 3.12 (t, J=7.2 Hz, 2H), 2.67 (t, J=7.7 Hz, 2H),2.48 (t, J=7.0 Hz, 2H), 1.94- 2.02 (m, 2H), 1.60-1.68 (m, 2H), 1.26-1.38(m, 14H), 0.92 (t, J=7.0 Hz, 3H). 50

384.2 51

382.2 ¹H NMR (500 MHz, CD₃OD) δ 8.30 (d, J=8.3 Hz, 2H), 7.65 (d, J=8.2Hz, 2H), 4.25 (s, 2H), 4.30 (s, 2H), 3.20 (t, J=7.3 Hz, 2H), 3.01 (t,J=7.2 Hz, 2H), 2.00-2.08 (m, 2H), 1.82-1.90 (m, 2H), 1.68-1.76 (m, 2H),1.48 (d, J=14.2 Hz, 3H), 1.26-1.44 (m, 12H), 0.92 (t, J=7.1 Hz, 3H). 52

364.1 53

396.2 ¹H NMR (500 MHz, CD₃OD) δ 7.77 (d, J=7.8 Hz, 1H), 7.42-7.43 (m,2H), 4.22 (s, 2H), 3.17 (t, J=7.3 Hz, 2H), 2.93 (t, J=7.3 Hz, 2H), 2.48(s, 3H), 1.96-2.06 (m, 2H), 1.82-1.88 (m, 2H), 1.64-1.70 (m, 2H), 1.47(d, J=14.0 Hz, 3H), 1.28-1.38 (m, 12H), 0.90 (t, J=7.0 Hz, 3H). 54

362.2 ¹H NMR (500 MHz, CD₃OD) δ 7.76 (d, J=8.4 Hz, 1H), 7.41-7.43 (m,2H), 4.23 (s, 2H), 3.14 (t, J=7.8 Hz, 2H), 2.93 (t, J=7.3 Hz, 2H), 2.48(t, J=7.0 Hz, 2H), 2.47 (s, 3H), 1.96-2.04 (m, 2H), 1.64-1.70 (m, 2H),1.26-1.40 (m, 12H), 0.91 (t, J=7.0 Hz, 3H). 55

398.2 ¹H NMR (500 MHz, CD₃OD) δ 7.76 (d, J=7.8 Hz, 1H), 7.42-7.43 (m,2H), 4.21 (s, 2H), 3.18 (t, J=7.2 Hz, 2H), 2.93 (t, J=7.3 Hz, 2H), 2.48(s, 3H), 1.98-2.08 (m, 2H), 1.80 (dt, J=18.1, 7.4 Hz, 2H), 1.64-1.71 (m,2H), 1.26-1.40 (m, 12H), 0.91 (t, J=7.0 Hz, 3H). 56

420.3 ¹H NMR (500 MHz, CD₃OD) δ 7.76 (d, J=8.3 Hz, 2H), 7.64 (s, 1H),7.59 (d, J=8.3 Hz, 2H), 7.55 (d, J=7.7 Hz, 1H), 7.45 (t, J=7.7 Hz, 1H),7.37 (d, J=7.6 Hz, 1H), 4.70 (t, 6.8 Hz, 1H), 4.27 (s, 2H), 3.21 (t,J=7.6 Hz, 2H), 2.00-2.10 (m, 2H), 1.70-1.88 (m, 4H), 1.26-1.50 (m, 8H),0.90 (t, J=7.0 Hz, 3H). 57

418.3 ¹H NMR (500 MHz, CD₃OD) δ 8.23 (s, 1H), 8.04 (d, J=7.7 Hz, 1H),7.91 (d, J=7.8 Hz, 1H), 7.80 (d, J=8.2 Hz, 2H), 7.62-7.66 (m, 3H), 4.28(s, 2H), 3.22 (t, 7.5 Hz, 2H), 3.11 (t, J=7.2 Hz, 2H), 2.02-2.12 (m,2H), 1.84 (dt, J=18.3, 7.4 Hz, 2H), 1.72-1.78 (m, 2H), 1.28-1.48 (m,6H), 0.94 (t, J=7.0 Hz, 3H). 58

468.2 ¹H NMR (500 MHz, CD₃OD) δ 7.29 (s, 1H), 7.16 (s, 1H), 4.01 (s,2H), 3.98 (t, J=6.4 Hz, 2H), 3.90 (s, 3H), 3.13 (t, J=6.7 Hz, 2H),1.98-2.01 (m, 2H), 1.73-1.77 (m, 4H), 1.49-1.51 (m, 2H), 1.32-1.34 (m,8H), 0.89-0.91 (m, 3H) 59

357.1 ¹H NMR (500 MHz, CD₃OD) δ 7.28 (s, 1H), 7.13 (s, 1H), 4.12-4.13(m, 2H), 4.09 (s, 3H), 4.00 (t, J=6.3, 2H), 3.12 (t, J=6.7, 2H),1.96-2.04 (m, 2H), 1.73-1.78 (m, 4H), 1.48-1.56 (m, 2H), 1.43-1.46 (m,2H), 1.32-1.37 (m, 8H), 0.88-0.93 (m, 3H) 60

436.2 ¹H NMR (500 MHz, CD₃OD) δ 7.7 (s, 1H), 7.41 (d, J=8.5 Hz, 1H),7.07 (d, J=8.4 Hz, 1H), 4.06–4.10 (m, 4H), 3.12 (t, J=7.2, 2H),1.95-2.00 (m, 2H), 1.75-1.83 (m, 4H), 1.51-1.54 (m, 2H), 1.32-1.37 (m,8H), 0.89-0.91 (m, 3H) 61

426.1 ¹H NMR (500 MHz, CD₃OD) δ 7.56 (s, 1H), 4.13 (s, 2H), 4.02-4.04(m, 2H), 3.13- 3.12 (m, 2H), 1.98-2.00 (m, 2H), 1.75-1.84 (m, 4H),1.49-1.58 (m, 2H), 1.26-1.42 (m, 8H), 0.89-0.91 (m, 3H) 62

386.3 ¹H NMR (500 MHz, CD₃OD) δ 7.14 (s, 2H), 4.08 (s, 2H), 3.79 (t,J=6.4 Hz, 2H), 3.13 (t, J=7.6 Hz, 2H), 2.30 (s, 6H), 1.95-2.05 (m, 2H),1.76-1.84 (m, 4H), 1.51-1.58 (m, 2H), 1.31-1.44 (m, 8H), 0.90-0.95 (m,3H) 63

364.2 ¹H NMR (500 MHz, CD₃OD) δ 7.40 (d, J=8.7 Hz, 2H), 7.26 (t, J=7.26Hz, 2H), 7.17-7.22 (m, 3H), 6.99 (d, J=8.7 Hz, 2H), 4.13 (s, 2H), 3.99(t, J=6.2 Hz, 2H), 3.13 (t, J=7.6 Hz, 2H), 2.81 (t, J=7.6 Hz, 2H),2.06-2.12 (m, 2H), 1.95-2.04 (m, 2H), 1.76-1.85 (m, 2H) 64

255.2 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.7 Hz, 2H), 7.26 (t, J=7.5Hz, 2H), 7.20 (d, J=7.1 Hz, 2H), 7.14-7.18 (m, 1H), 6.98 (d, J=8.7 Hz,2H), 4.12 (s, 2H), 4.02 (s, 2H), 3.12 (t, J=7.4 Hz, 2H), 2.66-2.72 (m,2H), 1.94-2.04 (m, 2H), 1.76-1.84 (m, 6H) 65

399.3 ¹H NMR (500 MHz, CD₃OD) δ 7.60 (d, J=7.8 Hz, 2H), 7.49 (t, J=7.3Hz, 2H), 4.26 (s, 2H), 3.19 (t, J=7.4 Hz, 3H), 3.09 (s, 2H), 2.96 (s,2H), 1.98-2.08 (m, 2H), 1.78-1.86 (m, 2H), 1.22-1.32 (m, 4H), 1.00-1.04(m, 8H)), 0.88-0.94 (m, 3H) 66

514.0 ¹H NMR (500 MHz, CD₃OD) δ 7.51 (s, 2H), 7.18 (d, 2H), 4.12 (s,2H), 3.99 (t, J=6.5 Hz, 2H), 3.90 (s, 3H), 3.15 (t, J=7.4 Hz, 2H),1.96-2.06 (m, 2H), 1.75-1.84 (m, 4H), 1.50-1.56 (m, 2H), 1.29-1.41 (m,8H), 0.89-0.95 (m, 3H) 67

462.1 ¹H NMR (500 MHz, CD₃OD) δ 6.99 (d, 2H), 4.14 (s, 2H), 3.97 (t,J=6.5 Hz, 2H), 3.89 (s, 3H), 3.15 (t, J=7.2 Hz, 2H), 2.93 (t, J=7.2 Hz,2H), 1.96-2.06 (m, 2H), 1.66- 1.84 (m, 6H), 1.48-1.56 (m, 2H), 1.28-1.42(m, 8H), 1.04-1.10 (m, 3H), 0.90-0.96 (m, 3H) 68

430.2 ¹H NMR (500 MHz, CD₃OD) δ 6.99 (s, 1H), 6.91 (s, 1H), 4.12 (s,2H), 3.95 (t, J=6.4 Hz, 2H), 3.88 (s, 3H), 3.15 (t, J=7.4 Hz, 2H), 2.62(t, J=7.8 Hz, 2H), 1.96-2.06 (m, 2H), 1.72-1.85 (m, 4H), 1.58-1.68 (m,2H), 1.48-1.54 (m, 2H), 1.30-1.42 (m, 8H), 0.95-1.00 (m, 3H), 0.90-0.95(m, 3H) 69

386.3 ¹H NMR (500 MHz, CD₃OD) δ 7.10 (s, 1H), 7.00 (s, 2H), 4.12 (s,2H), 4.02 (s, 2H), 3.89 (s, 3H), 3.10-3.16 (m, 2H), 1.94-2.04 (m, 2H),1.73-1.83 (m, 4H), 1.62-1.71 (m, 2H), 1.26-1.52 (m, 8H), 0.88-0.96 (m,3H) 70

422.1 ¹H NMR (500 MHz, CD₃OD) δ 7.16 (s, 1H), 7.13 (s, 1H), 4.13 (s,2H), 4.01 (t, J=6.6 Hz, 2H), 3.92 (s, 3H), 3.15 (t, J=7.2 Hz, 2H),1.96-2.06 (m, 2H), 1.72-1.84 (m, 4H), 1.48-1.55 (m, 2H), 1.28-1.41 (m,8H), 0.89-0.95 (m, 3H) 71

482.3 ¹H NMR (500 MHz, CD₃OD) δ 7.32 (d, 1H), 7.17 (d, 1H), 4.15 (s,2H), 4.01 (t, J=6.4 Hz, 2H), 3.92 (s, 3H), 3.17 (t, J=7.6 Hz, 2H),1.98-2.08 (m, 2H), 1.74-1.87 (m, 4H), 1.49-1.59 (m, 2H), 1.28-1.44 (m,10H), 0.90-0.96 (m, 3H) 72

454.2 ¹H NMR (500 MHz, CD₃OD) δ 7.32 (d, 1H), 7.17 (d, 1H), 4.15 (s,2H), 4.01 (t, J=6.5 Hz, 2H), 3.92 (s, 3H), 3.17 (t, J=7.5 Hz, 2H),1.98-2.08 (m, 2H), 1.75-1.86 (m, 4H), 1.49-1.56 (m, 2H), 1.32-1.43 (m,6H), 0.92-0.96 (m, 3H) 73

544.2 ¹H NMR (500 MHz, CD₃OD) δ 7.49 (d, J=7.3 Hz, 2H), 7.41 (t, J=7.4Hz, 2H), 7.37 (d, J=7.3 Hz, 1H), 7.33 (s, 1H), 7.25 (s, 1H), 5.18 (s,2H), 4.13 (s, 2H), 4.02 (t, J=6.4 Hz, 2H), 3.12 (t, J=7.3 Hz, 2H),1.96-2.06 (m, 2H), 1.70-1.84 (m, 4H), 1.40-1.48 (m, 2H), 1.22-1.36 (m,8H), 0.88-0.94 (m, 3H) 74

464.3 ¹H NMR (500 MHz, CD₃OD) δ 7.47 (d, J=7.5 Hz, 2H), 7.38 (t, J=7.4Hz, 2H), 7.33 (d, J=7.4 Hz, 1H), 7.15 (s, 1H), 7.04 (s, 2H), 5.16 (s,2H), 4.09 (s, 2H), 4.05 (t, J=6.3 Hz, 2H), 3.08 (t, J=7.4 Hz, 2H),1.93-2.04 (m, 2H), 1.73-1.84 (m, 4H), 1.47-1.55 (m, 8H), 1.26-1.41 (m,8H), 0.88-0.93 (m, 3H) 75

350.1 ¹H NMR (500 MHz, CD₃OD) δ 6.94-6.98 (m, 2H), 6.88-6.92 (m, 1H),4.05 (s, 4H), 3.32 (s, 2H), 3.11 (t, J=7.2 Hz, 2H), 1.94-2.04 (m, 2H),1.73-1.86 (m, 4H), 1.27-1.45 (m, 10H), 0.88-0.95 (m, 3H) 76

496.2 ¹H NMR (500 MHz, CD₃OD) δ 7.31 (s, 1H), 7.18 (s, 1H), 4.12 (s,2H), 4.00 (t, J=6.4 Hz, 2H), 3.92 (s, 3H), 3.15 (t, J=7.1 Hz, 2H),1.96–2.06 (m, 2H), 1.73–1.82 (m, 4H), 1.49–1.56 (m, 2H), 1.27–1.42 (m,12H), 0.89–0.94 (m, 3H) 77

438.0 ¹H NMR (500 MHz, CD₃OD) δ 7.31 (s, 1H), 7.18 (s, 1H), 4.12 (s,2H), 4.00 (t, J=6.4 Hz, 2H), 3.92 (s, 3H), 3.12–3.17 (t, 2H), 1.96-2.06(m, 2H), 1.73-1.82 (m, 4H), 1.48- 1.57 (m, 2H), 1.34-1.41 (m, 4H),0.91-0.97 (m, 3H) 78

510.1 ¹H NMR (500 MHz, CD₃OD) δ 7.31 (s, 1H), 7.18 (s, 1H), 4.11 (s,2H), 4.00 (t, J=6.4 Hz, 2H), 3.92 (s, 3H), 3.11-3.17 (t, 2H), 1.95-2.06(m, 2H), 1.71-1.81 (m, 4H), 1.48- 1.56 (m, 2H), 1.27-1.42 (m, 14H),0.88-0.94 (m, 3H) 79

302.1 ¹H NMR (500 MHz, CD₃OD) δ 7.31-7.40 (m, 2H), 7.02-7.08 (m, 2H),4.12 (s, 2H), 4.03 (t, J=6.4 Hz, 2H), 3.12 (t, J=6.4 Hz, 2H), 1.94-2.04(m, 2H), 1.66-1.81 (m, 4H), 1.48–1.56 (m, 2H), 0.97–1.02 (m, 3H) 80

442.2 ¹H NMR (500 MHz, CD₃OD) δ 7.43 (d, J=7.5 Hz, 4H), 7.38 (t, J=7.5Hz, 4H), 7.33 (d, J=7.1 Hz, 2H), 6.76 (s, 2H), 6.71 (s, 1H), 5.10 (s,4H), 4.08 (s, 2H), 3.08 (t, J=6.4 Hz, 2H), 1.93-2.04 (m, 2H), 1.68-1.76(m, 2H) 81

402.2 ¹H NMR (500 MHz, CD₃OD) δ 6.98 (s, 1H), 6.90 (s, 1H), 4.10 (s,2H), 3.94 (t, J=6.6 Hz, 2H), 3.88 (s, 3H), 3.14 (t, J=7.7 Hz, 2H), 2.27(s, 3H), 1.96-2.06 (m, 2H), 1.71- 1.85 (m, 4H), 1.46-1.54 (m, 2H),1.28-1.42 (m, 8H), 0.90-0.95 (m, 3H) 82

464.3 ¹H NMR (500 MHz, CD₃OD) δ 7.52 (d, J=7.4 Hz, 2H), 7.42 (t, J=7.4Hz, 2H), 7.36 (t, J=7.3 Hz, 1H), 7.17 (s, 1H), 7.08 (s, 1H), 4.20 (s,2H), 3.95 (s, 3H), 3.71 (t, J=6.3 Hz, 2H), 3.36 (s, 2H), 3.19 (t, J=7.5Hz, 2H), 1.98-2.09 (m, 2H), 1.78-1.87 (m, 2H), 1.41-1.48 (m, 2H),1.25-1.34 (m, 2H), 1.08-1.25 (m, 6H), 0.87-0.94 (m, 3H) 83

374.2 ¹H NMR (500 MHz, CD₃OD) δ 6.94-6.98 (m, 2H), 6.88-6.92 (m, 1H),4.05 (s, 4H), 3.32 (s, 2H), 3.11 (t, J=7.2 Hz, 2H), 1.94-2.04 (m, 2H),1.73-1.86 (m, 4H), 1.27-1.45 (m, 10H), 0.88-0.95 (m, 3H) 84

392.1 ¹H NMR (500 MHz, CD₃OD) δ 7.69 (d, J=8.0, 2H), 7.57 (d, J=8.7 Hz,2H), 7.54 (d, J=8.0 Hz, 2H), 7.01 (d, J=8.5 Hz, 2H), 4.22 (s, 2H), 4.03(t, 2H), 3.18 (t, 2H), 1.98- 2.08 (m, 2H), 1.76-1.86 (m, 4H), 1.40-1.53(m, 4H), 0.96-1.00 (m, 3H) 85

378.1 ¹H NMR (500 MHz, CD₃OD) δ 7.70 (d, J=8.3 Hz, 2H), 7.58 (d, J=8.7Hz, 2H), 7.55 (d, J=7.55 Hz, 2H), 7.02 (d, J=8.7 Hz, 2H), 4.24 (s, 2H),4.05 (t, J=6.4 Hz, 2H), 3.20 (t, J=7.6 Hz, 2H), 1.99-2.10 (m, 2H),1.76-1.88 (m, 4H), 1.51-1.59 (m, 2H), 1.00-1.08 (m, 3H) 86

406.2 ¹H NMR (500 MHz, CD₃OD) δ 7.70 (d, J=8.3 Hz, 2H), 7.58 (d, J=8.7Hz, 2H), 7.55 (d, J=8.3 Hz, 2H), 7.02 (d, J=8.4 Hz, 2H), 4.24 (s, 2H),4.04 (t, J=6.4 Hz, 2H), 3.16- 3.23 (t, 2H), 1.99-2.10 (m, 2H), 1.76-1.88(m, 4H), 1.48-1.58 (m, 2H), 1.36-1.45 (m, 4H), 0.91-1.00 (m, 3H) 87

468.3 ¹H NMR (500 MHz, CD₃OD) δ 7.69 (d, J=8.0 Hz, 2H), 7.67 (s, 1H),7.56 (d, J=8.2 Hz, 2H), 7.15 (d, J=8.5 Hz, 2H), 4.24 (s, 2H), 4.11 (t,J=6.1 Hz, 2H), 3.19 (t, J=7.2 Hz, 2H), 1.98-2.08 (m, 2H), 1.78-1.88 (m,4H), 1.51-1.59 (m, 2H), 1.29-1.46 (m, 8H), 0.88-0.96 (m, 3H) 88

434.1 ¹H NMR (500 MHz, CD₃OD) δ 7.68 (d, J=8.2 Hz, 2H), 7.54 (d, J=8.2Hz, 2H), 7.30 (s, 2H), 4.24 (s, 2H), 3.83 (t, J=6.5 Hz, 2H), 3.19 (t,J=7.4 Hz, 2H), 2.34 (s, 6H), 2.00- 2.09 (m, 2H), 1.78-1.88 (m, 4H),1.54-1.62 (m, 2H), 1.38-1.46 (m, 4H), 0.94-1.01 (m, 3H) 89

440.1 ¹H NMR (500 MHz, CD₃OD) δ 7.70 (d, J=8.0 Hz, 2H), 7.68 (s, 1H),7.57 (d, J=8.0 Hz, 3H), 7.16 (d, J=8.5 Hz, 1H), 4.25 (s, 2H), 4.12 (t,J=6.3 Hz, 2H), 3.20 (t, J=7.5 Hz, 2H), 2.00-2.09 (m, 2H), 1.80-1.90 (m,4H), 1.53-1.61 (m, 2H), 1.38-1.46 (m, 4H), 0.93-0.99 (m, 3H) 90

¹H NMR (500 MHz, CD₃OD) δ 7.57 (d, J=8.0 Hz, 2H), 7.24 (d, J=7.8 Hz,2H), 6.67 (s, 2H), 4.25 (s, 2H), 3.94-4.00 (t, 2H), 3.18-3.25 (t, 2H),2.00-2.05 (m, 2H), 1.99 (s, 6H), 1.78-1.90 (m, 4H), 1.45-1.55 (m, 2H),1.35-1.40 (m, 4H), 0.95-1.00 (m, 3H) 91

454.2 ¹H NMR (500 MHz, CD₃OD) δ 7.68 (d, J=8.0 Hz, 2H), 7.57 (d, J=7.57,2H), 7.51 (s, 1H), 7.43 (s, 1H), 4.22 (s, 2H), 3.97 (t, J=6.3 Hz, 2H),3.14-3.22 (t, 2H), 2.38 (s, 3H), 1.98-2.08 (m, 2H), 1.74-1.88 (m, 4H),1.54-1.62 (m, 2H), 1.36-1.46 (m, 4H), 0.92-1.00 (m, 3H) 92

436.3 ¹H NMR (500 MHz, CD₃OD) δ 7.71 (d, J=8.0 Hz, 2H), 7.54 (d, J=8.3Hz, 2H), 7.20- 7.23 (m, 1H), 7.18-7.20 (m, 1H), 7.04 (d, J=8.5 Hz, 1H),4.24 (s, 2H), 4.05 (t, J=6.5 Hz, 2H), 3.92 (s, 3H), 3.19 (t, J=7.4 Hz,2H), 2.00-2.08 (m, 2H), 1.78-1.88 (m, 4H), 1.48-1.56 (m, 2H), 1.36-1.43(m, 4H), 0.92-0.98 (m, 3H) 93

¹H NMR (500 MHz, CD₃OD) δ 7.71 (d, J=8.1 Hz, 2H), 7.57 (d, J=7.5 Hz,2H), 7.32- 7.39 (m, 1H), 7.10-7.21 (m, 2H), 6.90-6.96 (m, 1H), 4.16-4.25(m, 2H), 4.00-4.08 (m, 2H), 3.12-3.22 (m, 2H), 1.96-2.06 (m, 2H),1.72-1.84 (m, 2H), 1.62-1.72 (m, 2H), 1.50-1.60 (m, 2H), 1.38-1.48 (m,2H), 0.98-1.06 (m, 3H) 94

302.1 ¹H NMR (500 MHz, CD₃OD) δ 7.69-7.74 (m, 2H), 7.57 (d, J=7.6 Hz,2H), 7.32-7.39 (m, 1H), 7.19 (d, J=7.1 Hz, 1H), 7.15 (s, 1H), 6.94 (d,J=8.0 Hz, 1H), 4.25 (s, 2H), 4.03-4.05 (m, 2H), 3.18-3.21 (m, 2H),1.97-2.09 (m, 2H), 1.76-1.88 (m, 4H), 1.46-1.54 (m, 2H), 1.38-1.46 (m,2H), 0.94-1.00 (m, 3H) 95

406.1 ¹H NMR (500 MHz, CD₃OD) δ 7.73 (d, J=8.3 Hz, 2H), 7.58 (d, J=8.2Hz, 2H), 7.38 (t, J=7.9 Hz, 1H), 7.21 (d, J=7.8 Hz, 1H), 7.16 (s, 1H),6.90 (d, J=6.0 Hz, 1H), 4.26 (s, 2H), 4.05 (t, J=6.4 Hz, 2H), 3.21 (t,J=7.5 Hz, 2H), 2.00-2.10 (m, 2H), 1.78-1.88 (m, 4H), 1.50-1.56 (m, 2H),1.36-1.44 (m, 4H), 0.92-0.98 (m, 3H) 96

382.0 ¹H NMR (500 MHz, CD₃OD) δ 7.87 (s, 1H), 7.82 (d, J=7.7 Hz, 2H),7.69 (d, J=7.8 Hz, 2H), 7.59-7.67 (m, 4H), 7.54-7.59 (m, 1H), 7.49 (t,J=7.6 Hz, 2H), 7.36-7.42 (m, 1H), 4.29 (s, 2H), 3.22 (t, J=7.6 Hz, 2H),2.00-2.12 (m, 2H), 1.80-1.90 (m, 2H) 97

382.0 ¹H NMR (500 MHz, CD₃OD) δ 7.45-7.48 (m, 2H), 7.40-7.45 (m, 2H),7.36 (d, J=8.1 Hz, 2H), 7.25 (d, J=8.3 Hz, 2H), 7.18-7.22 (m, 3H),7.11-7.15 (m, 2H), 4.17 (s, 2H), 3.14 (t, J=7.6 Hz, 2H), 1.99-2.01 (m,2H), 1.95-1.97 (m, 2H) 98

420.3 ¹H NMR (500 MHz, CD₃OD) δ 7.72 (d, J=8.0 Hz, 2H), 7.57 (d, J=8.0Hz, 2H), 7.34- 7.39 (t, 1H), 7.18-7.22 (d, 1H), 7.15 (s, 1H), 6.92-6.96(d, 1H), 4.25 (s, 2H), 4.04 (t, J=6.4 Hz, 2H), 3.19 (t, J=7.5 Hz, 2H),1.98-2.08 (m, 2H), 1.76-1.86 (m, 4H), 1.47-1.55 (m, 2H), 1.31-1.45 (m,6H), 0.90-0.96 (m, 3H) 99

376.2 ¹H NMR (500 MHz, CD₃OD) δ 7.72 (d, J=8.3 Hz, 2H), 7.56 (d, J=8.0Hz, 4H), 7.29 (d, J=8.0 Hz, 2H), 4.24 (s, 2H), 3.19 (t, J=7.6 Hz, 2H),2.66 (t, J=7.6 Hz, 2H), 2.00- 2.09 (m, 2H), 1.79-1.87 (m, 2H), 1.63-1.70(m, 2H), 1.28-1.41 (m, 4H), 0.89-0.96 (m, 3H) 100

390.3 ¹H NMR (500 MHz, CD₃OD) δ 7.72 (d, J=8.0 Hz, 2H), 7.56 (d, J=7.8Hz, 4H), 7.29 (d, J=8.0 Hz, 2H), 4.25 (s, 2H), 3.19 (t, J=7.5 Ha, 2H),2.67 (t, J=7.7, 2H), 2.00-2.09 (m, 2H), 1.78-1.87 (m, 2H), 1.61-1.70 (m,2H), 1.31-1.41 (m, 6H), 0.98-0.94 (m, 3H) 101

404.2 ¹H NMR (500 MHz, CD₃OD) δ 7.73 (d, J=8.0 Hz, 2H), 7.57 (d, J=7.6Hz, 2H), 7.30 (d, J=8.3 Hz, 4H), 4.26 (s, 2H), 3.20 (t, J=7.6 Hz, 2H),2.68 (t, J=7.7 Hz, 2H), 2.00- 2.10 (m, 2H0, 1.80-1.88 (m, 2H), 1.64-1.70(m, 2H), 1.26-1.40 (m, 8H), 0.90-0.95 (m, 3H) 102

330.1 ¹H NMR (500 MHz, CD₃OD) δ 7.37 (t, J=8.0 Hz, 1H), 6.99-7.07 (m,3H), 4.16 (s, 2H), 4.02 (t, J=6.6 Hz, 2H), 3.15 (t, J=7.6 Hz, 2H),1.96-2.06 (m, 2H), 1.75-1.84 (m, 4H), 1.46-1.54 (m, 2H), 1.34-1.46 (m,8H), 0.91-0.97 (m, 3H) 103

416.3 ¹H NMR (500 MHz, CD₃OD) δ 7.07 (s, 1H), 6.99 (s, 2H), 4.09 (s,2H), 4.03 (t, J=6.3 Hz, 2H), 3.97 (t, J=6.3 Hz, 2H), 3.11 (J=7.1 Hz,2H), 1.93-2.04 (m, 2H), 1.72-1.84 (m, 6H), 1.46-1.54 (m, 2H), 1.26-1.42(m, 8H), 1.02-1.08 (m, 3H), 0.86-0.94 (m, 3H) 104

392.2 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.4 Hz, 2H), 7.25 (t, J=7.5Hz, 2H), 7.12- 7.19 (m, 3H), 6.98 (d, J=8.7 Hz, 2H), 4.12 (s, 2H), 4.00(t, J=6.4 Hz, 2H), 3.13 (t, J=7.5 Hz, 2H), 2.65 (t, J=7.6 Hz, 2H), 1.94(m, 2H), 1.74-1.86 (m, 4H), 1.66-1.74 (m, 2H), 1.48-1.56 (m, 2H) 105

408.4 ¹H NMR (500 MHz, CD₃OD) δ 7.91 (s, 1H), 7.86 (d, J=8.4 Hz, 1H),7.82 (d, J=8.9 Hz, 1H), 7.51 (d, J=8.5 Hz, 1H), 7.27 (s, 1H), 7.21 (d,J=8.8 Hz, 1H), 4.32 (s, 2H), 4.11 (t, J=6.3 Hz, 2H), 3.16-3.22 (m, 2H),1.98-2.08 (m, 2H), 1.76-1.90 (m, 4H), 1.48- 1.58 (m, 2H), 1.28-1.46 (m,8H), 0.90-0.96 (m, 3H) 106

440.4 107

426.3 ¹H NMR (500 MHz, CD₃OD) δ 7.71 (d, J=7.8 Hz, 2H), 7.56 (d, J=8.0,2H), 7.28- 7.39 (m, 5H), 7.18-7.25 (m, 2H), 7.14 (s, 1H), 6.95 (d, J=8.0Hz, 1H), 4.22–4.31 (m, 4H), 3.19 (d, J=7.4 Hz, 2H), 3.11 (d, J=6.6 Hz,2H), 1.97-2.09 (m, 2H), 1.78-1.88 (m, 2H)

Example 108 (R/S)-3-(N-(4-Nonylbenzyl)amino-1-hydroxypropylphosphonicacid Step A: (R/S)-Diethyl3-benzyloxycarbonylamino-1-hydroxypropylphosphonate

To a solution of potassium bis(trimethylsilyl)amide (1.13 g, 5.66 mmol)in tetrahydrofuran (10 mL) at 0° C. was added diethyl phosphite (0.73 g,5.66 mmol). After 10 min, 3-(benzyloxycarbonylamino)propanal (0.78 g,3.77 mmol) was added as a solution in tetrahydrofuran (5 mL). After 30min, the reaction was quenched by the addition of 2N hydrochloric acid(25 mL) and extracted with ethyl acetate (50 mL). The organic layer waswashed with sat'd sodium chloride (50 mL), dried over magnesium sulfateand concentrated in vacuo. Silica gel chromatography eluting withhexane/acetone (1:1) gave a colorless oil (0.36 g): ESI-MS 346.1 (M+H).

Step B: (R/S)-Diethyl 3-amino-1-hydroxypropylphosphonate

(R/S)-Diethyl 3-benzyloxycarbonylamino-1-hydroxypropylphosphonate (0.36g, 1.04 mmol, from Step A) and palladium on carbon (10%, 0.10 g) werestirred together in methanol (5 mL) under an atmosphere of hydrogen.After 2 h, the reaction was filtered and concentrated in vacuo to give acolorless oil: ¹H NMR (500 MHz, CD₃OD) δ 4.10-4.22 (m, 4H), 4.00-4.05(m, 1H), 2.85-3.00 (m, 2H), 1.85-2.00 (m, 2H), 1.34 (t, J=7.0 Hz, 6H);ESI-MS 211.8 (M+H)

Step: C (R/S)-Diethyl3-(N-(4-nonylbenzyl)amino-1-hydroxypropylphoslphonate

(R/S)-Diethyl 3-amino-1-hydroxypropylphosphonate (0.030 g, 0.142 mmol,from Step C), 4-nonylbenzaldehyde (0.036 g, 0.142 mmol) and sodiumcyanoborohydride (0.004 g, 0.071 mmol) in methanol (1.5 mL) were heatedat 50° C. for 3 h. The reaction was made acidic (pH˜5) by the additionof concentrated hydrochloric acid then directly purified by LC-3 to givea colorless oil (0.031 g).

Step D: (R/S)-3-(N-(4-nonylbenzyl)amino-1-hydroxypropylphosphonic acid

(R/S)-Diethyl 3-(N-(4-nonylbenzyl)amino-1-hydroxypropylphosphonate(0.031 g) was dissolved in acetonitrile (1 mL) and treated withbromotrimethylsilane (0.050 mL, 0.362 mmol). After stirring for 1 h at50° C., the reaction was quenched with methanol (1 mL), stirred for 30min then concentrated. The residue was purified via HPLC to give desiredproduct (0.011 g): ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.3 Hz, 2H),7.28 (d, J=8.3 Hz, 2H), 4.16 (s, 2H), 3.87-3.92 (m, 1H), 3.18-3.34 (m,2H), 2.64 (t, J=7.7 Hz, 2H), 2.04-2.20 (m, 2H), 1.58-1.64 (m, 2H),1.24-1.34 (m, 12H), 0.89 (t, J=7.0 Hz, 3H); ESI-MS 372.2 (M+H).

Examples 109-111

The following EXAMPLES (109-111) were made according to the proceduredescribed for EXAMPLE 108 substituting A for 4-nonylbenzaldehyde and thediethyl ester of B for (R/S)-diethyl 3-amino-1-hydroxyphosphonate inStep C.

EXAMPLE A B ESI-MS 109

372.1 ¹H NMR (500 MHz, CD₃OD) δ 7.42 (d, J=8.0 Hz, 2H), 7.31 (d, J=8.0Hz, 2H), 4.24- 4.50 (m, 1H, 4.21 (s, 2H), 3.30-3.38 (m, 1H), 3.01 (dd,J=12.8, 9.6 Hz, 1H, 2.67 (t, J=7.7 Hz, 2H), 1.94-2.14 (m, 2H), 1.60-1.68(m, 2H), 1.26-1.38 (m, 12H), 0.92 (t, J=7.0 Hz, 3H) 110

482.2 ¹H NMR (500 MHz, CD₃OD) δ 7.33 (d, J=1.9 Hz, 1H), 7.19 (d, J=1.8Hz, 1H, 4.22- 4.28 (m, 1H), 4.18 (s, 2H), 4.01 (t, J=6.4 Hz, 2H), 3.93(s, 3H), 3.30-3.35 (m, 1H), 3.03 (dd, J=12.6, 8.7 Hz, 1H), 1.91-2.11 (m,2H), 1.75-1.82 (m, 2H), 1.50-1.58 (m, 2H), 1.30-1.42 (m, 8H), 0.93 (t,J=7.0 Hz, 3H) 111

482.1 ¹H NMR (500 MHz, CD₃OD) δ 7.33 (d, J=2.1 Hz, 1H), 7.19 (d, J=1.8Hz, 1H), 4.18 (s, 2H), 4.02 (t, J=6.4 Hz, 2H), 3.92-3.96 (m, 1H), 3.93(s, 3H), 3.23-3.36 (m, 2H), 2.08-2.26 (m, 2H), 1.75-1.82 (m, 2H),1.50-1.58 (m, 2H), 1.30-1.42 (m, 8H), 0.93 (t, J=7.0 Hz, 3H)

Example 112 N-(4-Nonylbenzyl)-3-aminopropylphosphonic acid

3-Aminopropylphosphonic acid (0.060 g, 0.436 mmol) andtetrabutylammonium hydroxide (1.0M in methanol, 0.44 mL, 0.43 mmol) inmethanol (3 mL) were heated at 50° C. for 15 min until all of the solidshad dissolved. 4-(Nonyl)benzyliodide (0.100 g, 0.291 mmol) and DIEA(0.112 g, 0.872 mmol) were added and stirring was continued for 12 h at50° C. The reaction was made acidic (pH˜5) by the addition ofconcentrated hydrochloric acid then directly purified using LC-3 to givethe title compound (0.020 g): ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J=8.0Hz, 2H), 7.29 (d, J=8.0 Hz, 2H), 4.15 (s, 2H), 3.14 (t, J=7.6 Hz, 2H),2.64 (t, J=7.7 Hz, 2H), 2.00 (m, 2H), 1.79 (td, J=5.3, 18.5 Hz, 2H),1.61 (m, 2H), 1.24-1.36 (m, 14H), 0.89 (t, J=7.0 Hz, 3H); ESI-MS 356.2(M+H).

Example 113 3-[(4-Octylbenzyl)amino]propylphosphinic acid Step A: Ethyl2-cyanoethyl(diethoxymethyl)phosphinate

To a solution 2.6234 g (13.37 mmol) of ethyldiethoxymethyl phosphinatein 10 mL EtOH was added 0.5670 g (10.70 mmol) acrylonitrile. Theresulting mixture was added to a solution of 0.071 g (2.81 mmol) NaH in10 mL EtOH at 0° C. The ice bath was removed at the end of the addition,and the reaction mixture was stirred at rt for 16 hr. The mixture wasneutralized (pH=7) with HOAc, and was partitioned between EtOAc and H₂O.The organic layer was separated, dried and concentrated, which provided2.47 g (93% yield) of the title compound: ¹H NMR (500 MHz) δ 1.25 (t,J=6.9, 6H), 1.34 (t, J=7.1, 3H), 2.11-2.19 (m, 2H), 2.68-2.74 (m, 2H),3.62-3.73 (m, 2H), 3.80-3.87 (m, 2H), 4.13-4.25 (m, 2H), 4.70 (d, J=6.4,1H); ESI-MS 250 (M+H).

Step B: Ethyl 3-Aminopropyl(diethoxymethyl)phosphinate

To a solution of 2.47 g (9.91 mmol) of ethyl 2-cyanoethyl(diethoxymethyl)phosphinate (from Step A) in 20 mL 2.0 M ammonia in EtOHwas added 250 mg Raney Nickel. The mixture was subjected tohydrogenation conditions (H₂, 40 psi, rt) for 16 hr. The reactionmixture was filtered over Celite and partitioned between CH₂Cl₂ and H₂O.The aqueous phase was extracted twice with CH₂Cl₂. The organic layer andextractions were combined, dried, and concentrated to provide 2.13 g(85% yield) of the title compound: ¹H NMR (500 MHz) δ 1.23 (dt, J₁=7.1,J₂=1.6 6H), 1.29 (t, J=7.1, 3H), 1.42 (s, br, 2H), 1.71-1.82 (m, 4H),2.72-2.75 (m, 2H), 3.63-3.70 (m, 2H), 3.78-3.86 (m, 2H), 4.08-4.21 (m,2H), 4.64 (d, J=6.7, 1H); ESI-MS 254 (M+H).

Step C: 3-[(4-Octylbenzyl)amino]propylphosphinic acid

A mixture of 98.5 mg (0.389 mmol) of ethyl 3-aminopropyl(diethoxymethyl)phosphinate (from Step B) and 84.9 mg (0.389 mmol) of4-octylbenzaldehyde in 1 mL of MeOH at rt was treated with 12.2 mg(0.194 mmol) Na(CN)BH₃. The resulting reaction mixture was stirred at rtfor 16 hr. The reaction was quenched with 0.5 mL of 12 N HCl, thenheated up to 80° C. for 1 hr. The mixture was cooled and concentrated.HPLC purification (LC-2) afforded 60 mg (47%) of the title compound: ¹HNMR (500 MHz, CD₃OD) δ 0.88 (t, J=7.1, 3H), 1.25-1.33 (m, 10H),1.59-1.66 (m, 4H), 1.90-1.96 (m, 2H), 2.63 (t, J=7.7, 2H), 3.09 (t,J=6.9, 2H), 4.12 (s, 2H), 7.03 (d, J=505.6, 1H), 7.27 (d, J=8.0, 2H),7.38 (d, J=8.0, 2H); LC-1: 3.02 min; ESI-MS 326 (M+H).

Examples 114-116

The following compounds were prepared using procedures analogous tothose described in EXAMPLE 113 substituting the appropriate Aldehyde for4-octylbenzaldehyde in Step C.

LC-1 ESI-MS EXAMPLE R (min) (M + H) 114 CH₃(CH₂)₈— 3.00 340 115CH₃(CH₂)₈O— 2.93 356 116 CH₃(CH₂)₉— 3.23 354

Example 117 3-(N-(4-(4′-Pentyl)biphenylmethyl))aminopropylphosphinicacid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting Aldehyde 56 for 4-octylbenzaldehyde in Step C:LC-1: 2.86 min; ESI-MS 360 (M+H).

Example 118 3-(N-(4-(4′-Heptyloxy)biphenylmethyl))aminopropylphosphinicacid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting Aldehyde 51 for 4-octylbenzaldehyde in Step C:LC-1: 3.06 min; ESI-MS 404 (M+H).

Example 1193-N-(3-Bromo-5-methoxy-4-(octyloxy)benzyl)aminopropylphosphinic acid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting Aldehyde 13 for 4-octylbenzaldehyde in Step C:LC-1: 2.98 min; ESI-MS 450 (M+H).

Example 120 3-N-(3-Fluoro-4-(nonyloxybenzyl)aminopropylphosphinic acid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting 3-fluoro-4-(nonyloxy)benzaldehyde for4-octylbenzaldehyde in Step C: ¹H NMR (500 MHz) δ 0.91 (t, J=7.0, 3H),1.30-1.40 (m, 10H), 1.48-1.51 (m, 2H), 1.71-1.99 (m, 6H), 3.11 (t,J=7.2, 2H), 4.07 (t, J=6.4, 2H), 4.12 (s, 2H), 7.06 (d, J=519, 1H),7.13-7.29 (m, 3H); LC-1: 2.96 min; ESI-MS 374 (M+H).

Example 121 3-N-(2-Chloro-4-(nonyloxy)benzyl)aminopropylphosphinic acid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting 2-chloro-4-(nonyloxy)benzaldehyde for4-octylbenzaldehyde in Step C: LC-1: 3.07 min; ESI-MS 390 (M+H).

Example 122 3-N-(6-Heptyloxy)napthylmethyl)aminopropylphosphinic acid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting 6-heptyloxy-1-napthaldehyde for4-octylbenzaldehyde in Step C: LC-1: 2.90 min; ESI-MS 378 (M+H).

Example 1233-(N-(3-Cyclopropyloxy-4-(nonyloxy)benzyl)amino)propylphosphinic acid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting Aldehyde 77 for 4-octylbenzaldehyde in Step C:LC-1: 3.04 min; ESI-MS 412 (M+H).

Example 124 3-(N-(4-(Nonylthio)benzyl)amino)propylphosphinic acid

The title compound was using a procedure analogous to that described inEXAMPLE 113, substituting Aldehyde 78 for 4-octylbenzaldehyde in Step C:¹H NMR (500 MHz) (CD₃OD) δ 0.90 (t, J=7.0, 3H), 1.30-1.32 (m, 10H),1.43-1.46 (m, 2H), 1.63-1.66 (m, 2H), 1.78-1.83 (m, 2H), 1.95-1.99 (m,2H), 2.98 (t, J=7.2, 2H), 3.14 (t, J=7.5, 2H), 4.16 (s, 2H), 7.08 (d,J=533, 1H), 7.37-7.42 (m, 4H); LC-1: 3.10 min; ESI-MS 372 (M+H).

Example 125 Ethyl(3-(4-nonylbenzyl)amino)propylphosphinic acid

A solution of 88 mg (0.26 mmol) of3-((4-nonylbenzyl)amino)propylphosphinic acid (from EXAMPLE 114) in 1 mLN,N-bis(trimethylsilyl)amine was heated to 100° C. for 8 hr. Uponcooling to rt, 81.1 mg (0.52 mmol) of iodoethane was added, followed bythe addition of 67.2 mg (0.52 mmol) of DIEA. The resulting mixture washeated to 60° C. overnight. The reaction mixture was cooled andconcentrated. HPLC purification (LC-2) afforded 12 mg (13%) of the titlecompound. ¹H NMR (500 MHz) (CD₃OD) δ 0.88 (t, J=7.1, 3H), 1.09-1.18 (m,3H), 1.26-1.31 (m, 12H), 1.59-1.75 (m, 6H), 1.94-2.00 (m, 2H), 2.63 (t,J=7.6, 2H), 3.10 (t, J=6.9, 2H), 4.13 (s, 2H), 7.27 (d, J=8.0, 2H), 7.39(d, J=8.0 2H); LC-1: 2.92 min; ESI-MS 368 (M+H).

Examples 126-127

The following compounds were prepared a procedure analogous to thatdescribed in EXAMPLE 125 substituting the appropriate alkyl halide forethyl iodide.

LC-1 ESI-MS EXAMPLE R (min) (M + H) 126 CH₃CH₂CH₂— 3.03 382 127 PhCH₂—3.41 430

Example 128 Hydroxymethyl(3-(4-nonylbenzyl)amino)propylphosphinic acid

A solution of 71 mg (0.21 mmol) of3-(4-nonylbenzyl)aminopropylphosphinic acid (from EXAMPLE 114) in 1 mLof N,N-(trimethylsilyl)amine was heated to 100° C. for 8 hr. Uponcooling to rt, 15.8 mg (0.53 mmol) of paraformaldehyde was added. Theresulting mixture was heated at 30° C. for 3 hr, and stirred at rt undernitrogen for 16 hr. The reaction mixture concentrated. HPLC purification(LC-2) afforded 22 mg (28%) of the title compound. ¹H NMR (500 MHz)(CD₃OD) δ 0.88 (t, J=7.1, 3H), 1.27-1.31 (m, 12H), 1.57-1.63 (m, 2H),1.80-1.85 (m, 2H), 1.97-2.05 (m, 2H), 2.63 (t, J=7.8, 2H), 3.12 (t,J=6.9, 2H), 3.70 (d, J=6.2, 2H), 4.13 (s, 2H), 7.27 (d, J=8.0, 2H), 7.39(d, J=8.2, 2H); LC-1: 2.90 min; ESI-MS 370 (M+H).

Example 129-133

The following compounds were prepared using a procedure analogous tothat described in EXAMPLE 128 substituting the appropriate aldehyde forparaformaldehyde.

LC-1 ESI-MS EXAMPLE R (min) (M + H) 129 CH₃— 2.89 384 130 CH₃CH₂— 2.95398 131

3.26 446 132

3.25 482 133

3.45 514

Example 134 Hydroxymethyl(3-(4-octylbenzyl)amino)propylphosphinic acid

The title compound was prepared from3-(4-octylbenzyl)aminopropylphosphinic acid (from EXAMPLE 114) using aprocedure analogous to that described in EXAMPLE 128: LC-1: 2.67 min;ESI-MS 356 (M+H).

Example 135 Hydroxymethyl3-(3-(cyclopropyloxy)-4-(nonyloxy)benzyl)aminopropylphosphinic acid

The title compound was prepared from3-(3-(cyclopropyloxy)-4-(nonyloxy)benzyl)aminopropylphosphinic acid(from EXAMPLE 123) using a procedure analogous to that described inEXAMPLE 128: LC-1: 2.95 min; ESI-MS 442 (M+H).

Example 136 Hydroxymethyl3-(3-fluoro-4-(nonyloxy)benzyl)aminopropylphosphinic acid

The title compound was prepared from3-(3-fluoro-4-(nonyloxy)benzyl)amino-propylphosphinic acid (from EXAMPLE125) using a procedure analogous to that described in EXAMPLE 128: LC-1:2.87 min; ESI-MS 404 (M+H).

Example 137 Ethoxycarbonyl3-(N-(4-(4′-heptyloxy)biphenylmethyl))aminopropylphosphinic acid

To a solution of 32.5 mg (0.081 mmol) of3-(N-(4-(4′-heptyloxy)biphenylmethyl))aminopropylphosphinic acid (fromEXAMPLE 118) in 2 mL dichloromethane was added 0.1 mL of TMSCl and 0.12mL of DIEA at 0° C. The solution was stirred at rt for an additional onehour and 0.1 mL of ethyl chloroformate (0.81 mmol) was added. Thereaction was quenched with MeOH and concentrated to oil. The product wasisolated and purified by LC-2: ¹H NMR (500 MHz) (CD₃OD) δ 0.94 (t,J=6.9, 3H), 1.31-1.43 (m, 8H), 1.51-1.53 (m, 2H), 1.80-1.83 (m, 2H),1.89-1.92 (m, 2H), 2.03-2.06 (m, 2H), 3.18 (t, J=6.7, 2H), 4.05 (t,J=6.4, 2H), 4.24 (s, 2H), 4.25 (q, J=7.0, 2H), 6.95-7.72 (m, 8H); LC-1:3.26 min; ESI-MS 476 (M+H).

Example 138 3-(4-Octylbenzyl)amino-2-phenylpropylphosphinic acid

A mixture of 69.2 mg (0.210 mmol) of ethyl3-amino-2-phenylpropyl(diethoxymethyl)phosphinate (Tetrahedron, 1989,3787-3808) and 48.2 mg (0.221 mmol) of 4-octylbenzaldehyde in 1 mL ofMeOH at rt was treated with 6.7 mg (0.105 mmol) of Na(CN)BH₃. Theresulting reaction mixture was stirred at rt for 16 hr. The reaction wasquenched with 0.3 mL of 12 N HCl, then heated up to 60° C. for 5 hr. Themixture was cooled and concentrated. HPLC purification (LC-2) afforded22 mg (26%) of the title compound. ¹H NMR (500 MHz) (CD₃OD) δ 0.88 (t,J=7.1, 3H), 1.26-1.30 (m, 10H), 1.58-1.61 (m, 2H), 2.01-2.17 (m, 2H),2.62 (t, J=7.8, 2H), 3.20-3.23 (m, 1H),3.35-3.46 (m, 2H), 4.11 (s, 2H),6.92 (d, J=535.4, 1H), 7.23-7.37 (m, 9H); LC-1: 3.31 min; ESI-MS 402(M+H).

Example 1393-(3-Bromo-5-methoxy-4-(octyloxy)benzyl)amino-2-phenylpropylphosphinicacid

The title compound was prepared using a procedure analogous to thatdescribed in EXAMPLE 138 substituting Aldehyde 13 for4-octylbenzaldehyde: LC-1: 3.51 min; ESI-MS 526 (M+H).

Examples 140-150

The following compounds were prepared using a procedure analogous tothat described in EXAMPLE 1 substituting the appropriateaminoalkylcarboxylic acid or aminoalkylphosphonic acid for3-aminopropylphosphonic acid and either Aldehyde 79 or 80 for4-(decyloxy)benzaldehyde. The products were purified using LC-2.

LC-1 ESI-MS EXAMPLE X Y (min) (M + H) 140

—(CH₂)₃PO₃H₂ 3.01 524 141

—(CH₂)₃CO₂H 3.07 448 142 —CH₂O— —(CH₂)₃PO₃H₂ 2.77 486 143 —CH₂O——(CH₂)₃CO₂H 2.79 450 144 —CH₂O— —(CH₂)₂CO₂H 2.72 436 145 —CH₂O——CH₂CH(CH₃)CO₂H 3.00 450 146 —CH₂O— —CH₂CH(OH)CO₂H 147 —CH₂O——CH(n-Pr)CH₂CO₂H 3.11 478 ¹H NMR (500 MHz, CD₃OD) δ 0.97 (3H, t, J=7.3);1.29-1.51 (2H, m); 1.63-1.71 (1H, m); 1.78-1.84 (1H, m); 2.66-2.83 (3H,m); 3.46-3.54 (1H, m); 4.23 (2H, s); 5.38 (2H, s); 7.12 (2H, d, J=8.5);7.21 (1H, s); 7.41-7.44 (5H, m); 7.47 (2H, d, J=8.5) 148 —CH₂O——CH(i-Pr)CH₂CO₂H 3.06 478 ¹H NMR (500 MHz, CD₃OD) δ 0.97 (3H, d, J=6.8);1.01 (3H, d, J=6.8); 2.15-2.21 (1H, m); 2.66-2.83 (3H, m); 3.48-3.51(1H, m); 4.28 (2H, q, J=13 & 28); 5.39 (2H, s); 7.13 (2H, d, J=8.5);7.21 (1H, s); 7.42-7.47 (5H, m); 7.49 (2H, d, J=8.5) 149 —CH₂O——CH(CH₃)CH₂CO₂H 2.90 450 ¹H NMR (500 Mhz, CD₃OD) δ 1.42 (3H, d, J=6.6);2.66-2.79 (2H, m); 2.83 (1H, s); 3.59-3.64 (1H, m); 4.21 (2H, q, J=13 &28); 5.38 (2H, s); 7.13 (2H, d, J=8.4); 7.21 (1H, s); 7.42-7.45 (5H, m);7.47 (2H, d, J=8.4) 150 —CH₂O— —(CH₂)₄CO₂H 2.95 464 ¹H NMR (500 MHz,CD₃OD) δ 1.60-1.80 (4H, m); 2.30-2.50 (2H, m); 3.24 (2H, s); 4.53 (2H,s); 5.31 (2H, s); 7.13 (2H, d, J=8.4); 7.21 (1H, s); 7.42-7.45 (5H, m);7.47 (2H, d, J=8.4)Biological Activity

The S1P₁/Edg1, S1P₃/Edg3, S1P₂/Edg5, S1P₄/Edg6 or S1P₅/Edg8 activity ofthe compounds of the present invention can be evaluated using thefollowing assays:

Ligand Binding to Edg/S1P Receptors Assay

³³P-sphingosine-1-phosphate was synthesized enzymatically from γ³³P-ATPand sphingosine using a crude yeast extract with sphingosine kinaseactivity in a reaction mix containing 50 mM KH₂PO₄, 1 mMmercaptoethanol, 1 mM Na₃VO₄, 25 mM KF, 2 mM semicarbazide, 1 mMNa₂EDTA, 5 mM MgCl₂, 50 mM sphingosine, 0.1% TritonX-114, and 1 mCiγ³³P-ATP (NEN; specific activity 3000 Ci/mmol). Reaction products wereextracted with butanol and ³³P-sphingosine-1-phosphate was purified byHPLC.

Cells expressing EDG/S1P receptors were harvested with enzyme-freedissociation solution (Specialty Media, Lavallette, N.J.). They werewashed once in cold PBS and suspended in binding assay buffer consistingof 50 mM HEPES-Na, pH 7.5, 5 mM MgCl₂, 1 mM CaCl₂, and 0.5% fattyacid-free BSA. ³³P-sphingosine-1-phosphate was sonicated with 0.1 nMsphingosine-1-phosphate in binding assay buffer; 100 μl of the ligandmixture was added to 100 μl cells (1×10⁶ cells/mL) in a 96 wellmicrotiter dish. Binding was performed for 60 min at room temperaturewith gentle mixing. Cells were then collected onto GF/B filter plateswith a Packard Filtermate Universal Harvester. After drying the filterplates for 30 min, 40 μl of Microscint 20 was added to each well andbinding was measured on a Wallac Microbeta Scintillation Counter.Non-specific binding was defined as the amount of radioactivityremaining in the presence of 0.5 μM cold sphingosine-1-phosphate.

Alternatively, ligand binding assays were performed on membranesprepared from cells expressing Edg/S1P receptors. Cells were harvestedwith enzyme-free dissociation solution and washed once in cold PBS.Cells were disrupted by homogenization in ice cold 20 mM HEPES pH 7.4,10 mM EDTA using a Kinematica polytron (setting 5, for 10 seconds).Homogenates were centrifuged at 48,000×g for 15 min at 4° C. and thepellet was suspended in 20 mM HEPES pH 7.4, 0.1 mM EDTA. Following asecond centrifugation, the final pellet was suspended in 20 mM HEPES pH7.4, 100 mM NaCl, 10 mM MgCl₂. Ligand binding assays were performed asdescribed above, using 0.5 to 2 μg of membrane protein.

Agonists and antagonists of Edg/S1P receptors can be identified in the³³P-sphingosine-1-phosphate binding assay. Compounds diluted in DMSO,methanol, or other solvent, were mixed with probe containing³³P-sphingosine-1-phosphate and binding assay buffer in microtiterdishes. Membranes prepared from cells expressing Edg/S1P receptors wereadded, and binding to ³³P-sphingosine-1-phosphate was performed asdescribed. Determination of the amount of binding in the presence ofvarying concentrations of compound and analysis of the data bynon-linear regression software such as MRLCalc (Merck ResearchLaboratories) or PRISM (GraphPad Software) was used to measure theaffinity of compounds for the receptor. Selectivity of compounds forEdg/S1P receptors was determined by measuring the level of³³P-sphingosine-1-phosphate binding in the presence of the compoundusing membranes prepared from cells transfected with each respectivereceptor (S1P₁/Edg1, S1P₃/Edg3, S1P₂/Edg5, S1P₄/Edg6, S1P₅/Edg8).

³⁵S-GTPγS Binding Assay

Functional coupling of S1P/Edg receptors to G proteins was measured in a³⁵S-GTPγS binding assay. Membranes prepared as described in the LigandBinding to Edg/S1P Receptors Assay (1-10 μg of membrane protein) wereincubated in a 200 μl volume containing 20 mM HEPES pH 7.4, 100 mM NaCl,10 MM MgCl₂, 5 μM GDP, 0.1% fatty acid-free BSA (Sigma, catalog A8806),various concentrations of sphingosine-1-phosphate, and 125 pM ³⁵S-GTPγS(NEN; specific activity 1250 Ci/mmol) in 96 well microtiter dishes.Binding was performed for 1 hour at room temperature with gentle mixing,and terminated by harvesting the membranes onto GF/B filter plates witha Packard Filtermate Universal Harvester. After drying the filter platesfor 30 min, 40 μl of Microscint 20 was added to each well and bindingwas measured on a Wallac Microbeta Scintillation Counter.

Agonists and antagonists of S1P/Edg receptors can be discriminated inthe ³⁵S-GTPγS binding assay. Compounds diluted in DMSO, methanol, orother solvent, were added to microtiter dishes to provide final assayconcentrations of 0.01 nM to 10 μM. Membranes prepared from cellsexpressing S1P/Edg receptors were added, and binding to ³⁵S-GTPγS wasperformed as described. When assayed in the absence of the naturalligand or other known agonist, compounds that stimulate 35S-GTPγSbinding above the endogenous level were considered agonists, whilecompounds that inhibit the endogenous level of ³⁵S-GTPγS binding wereconsidered inverse agonists. Antagonists were detected in a ³⁵S-GTPγSbinding assay in the presence of a sub-maximal level of natural ligandor known S1P/Edg receptor agonist, where the compounds reduced the levelof ³⁵S-GTPγS binding. Determination of the amount of binding in thepresence of varying concentrations of compound was used to measure thepotency of compounds as agonists, inverse agonists, or antagonists ofS1P/Edg receptors. To evaluate agonists, percent stimulation over basalwas calculated as binding in the presence of compound divided by bindingin the absence of ligand, multiplied by 100. Dose response curves wereplotted using a non-linear regression curve fitting program MRLCalc(Merck Research Laboratories), and EC₅₀ values were defined to be theconcentration of agonist required to give 50% of its own maximalstimulation. Selectivity of compounds for S1P/Edg receptors wasdetermined by measuring the level of ³⁵S-GTPγS binding in the presenceof compound using membranes prepared from cells transfected with eachrespective receptor.

Intracellular Calcium Flux Assay

Functional coupling of S1P/Edg receptors to G protein associatedintracellular calcium mobilization was measured using FLIPR(Fluorescence Imaging Plate Reader, Molecular Devices). Cells expressingS1P/Edg receptors were harvested and washed once with assay buffer(Hanks Buffered Saline Solution (BRL) containing 20 mM HEPES, 0.1% BSAand 710 μg/mL probenicid (Sigma)). Cells were labeled in the same buffercontaining 500 nM of the calcium sensitive dye Fluo-4 (Molecular Probes)for 1 hour at 37° C. and 5% CO₂. The cells were washed twice with bufferbefore plating 1.5×10⁵ per well (90 μl) in 96 well polylysine coatedblack microtiter dishes. A 96-well ligand plate was prepared by dilutingsphingosine-1-phosphate or other agonists into 200 μl of assay buffer togive a concentration that was 2-fold the final test concentration. Theligand plate and the cell plate were loaded into the FLIPR instrumentfor analysis. Plates were equilibrated to 37° C. The assay was initiatedby transferring an equal volume of ligand to the cell plate and thecalcium flux was recorded over a 3 min interval. Cellular response wasquantitated as area (sum) or maximal peak height (max). Agonists wereevaluated in the absence of natural ligand by dilution of compounds intothe appropriate solvent and transfer to the Fluo-4 labeled cells.Antagonists were evaluated by pretreating Fluo-4 labeled cells withvarying concentrations of compounds for 15 min prior to the initiationof calcium flux by addition of the natural ligand or other S1P/Edgreceptor agonist.

Preparation of Cells Expressing S1P/Edg Receptors

Any of a variety of procedures may be used to clone S1P₁/Edg1,S1P₃/Edg3, S1P₂/Edg5, S1P₄/Edg6 or S1P₅/Edg8. These methods include, butare not limited to, (1) a RACE PCR cloning technique (Frohman, et al.,1988, Proc. Natl. Acad. Sci. USA 85: 8998-9002). 5′ and/or 3′ RACE maybe performed to generate a full-length cDNA sequence; (2) directfunctional expression of the Edg/S1P cDNA following the construction ofan S1P/Edg-containing cDNA library in an appropriate expression vectorsystem; (3) screening an S1P/Edg-containing cDNA library constructed ina bacteriophage or plasmid shuttle vector with a labeled degenerateoligonucleotide probe designed from the amino acid sequence of theS1P/Edg protein; (4) screening an S1P/Edg-containing cDNA libraryconstructed in a bacteriophage or plasmid shuttle vector with a partialcDNA encoding the S1P/Edg protein. This partial cDNA is obtained by thespecific PCR amplification of S1P/Edg DNA fragments through the designof degenerate oligonucleotide primers from the amino acid sequence knownfor other proteins which are related to the S1P/Edg protein; (5)screening an S1P/Edg-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA oroligonucleotide with homology to a mammalian S1P/Edg protein. Thisstrategy may also involve using gene-specific oligonucleotide primersfor PCR amplification of S1P/Edg cDNA; or (6) designing 5′ and 3′ genespecific oligonucleotides using the S1P/Edg nucleotide sequence as atemplate so that either the full-length cDNA may be generated by knownRACE techniques, or a portion of the coding region may be generated bythese same known RACE techniques to generate and isolate a portion ofthe coding region to use as a probe to screen one of numerous types ofcDNA and/or genomic libraries in order to isolate a full-length versionof the nucleotide sequence encoding S1P/Edg.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cell types-orspecies types, may be useful for isolating an S1P/Edg-encoding DNA or anS1P/Edg homologue. Other types of libraries include, but are not limitedto, cDNA libraries derived from other cells.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have S1P/Edgactivity. The selection of cells or cell lines for use in preparing acDNA library to isolate a cDNA encoding S1P/Edg may be done by firstmeasuring cell-associated S1P/Edg activity using any known assayavailable for such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

An expression vector containing DNA encoding an S1P/Edg-like protein maybe used for expression of S1P/Edg in a recombinant host cell. Suchrecombinant host cells can be cultured under suitable conditions toproduce S1P/Edg or a biologically equivalent form. Expression vectorsmay include, but are not limited to, cloning vectors, modified cloningvectors, specifically designed plasmids or viruses. Commerciallyavailable mammalian expression vectors may be suitable for recombinantS1P/Edg expression.

Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to, bacteria such as E. coli, fungal cells such as yeast,mammalian cells including, but not limited to, cell lines of bovine,porcine, monkey and rodent origin; and insect cells including but notlimited to Drosophila and silkworm derived cell lines.

The nucleotide sequences for the various S1P/Edg receptors are known inthe art. See, for example, the following:

S1P₁/Edg1 Human

Hla, T. and T. Maciag 1990 An abundant transcript induced indifferentiating human endothelial cells encodes a polypeptide withstructural similarities to G-protein coupled receptors. J. Biol Chem.265:9308-9313, hereby incorporated by reference in its entirety.

WO91/15583, published on Oct. 17, 1991, hereby incorporated by referencein its entirety.

WO99/46277, published on Sep. 16, 1999, hereby incorporated by referencein its entirety.

S1P₁/Edg1 Mouse

WO0059529, published Oct. 12, 2000, hereby incorporated by reference inits entirety.

U.S. Pat. No. 6,323,333, granted Nov. 27, 2001, hereby incorporated byreference in its entirety.

S1P₁/Edg1 Rat

Lado, D. C., C. S. Browe, A. A. Gaskin, J. M. Borden, and A. J.MacLennan. 1994 Cloning of the rat edg-1 immediate-early gene:expression pattern suggests diverse functions. Gene 149: 331-336, herebyincorporated by reference in its entirety.

U.S. Pat. No. 5,585,476, granted Dec. 17, 1996, hereby incorporated byreference in its entirety.

U.S. Pat. No. 5,856,443, granted Jan. 5, 1999, hereby incorporated byreference in its entirety.

S1P₃/Edg3 Human

An, S., T. Bleu, W. Huang, O. G. Hallmark, S. R. Coughlin, E. J. Goetzl1997 Identification of cDNAs encoding two G protein-coupled receptorsfor lysosphingolipids FEBS Lett. 417:279-282, hereby incorporated byreference in its entirety.

WO 99/60019, published Nov. 25, 1999, hereby incorporated by referencein its entirety.

U.S. Pat. No. 6,130,067, granted Oct. 10, 2000, hereby incorporated byreference in its entirety.

S1P₃/Edg3 Mouse

WO 01/11022, published Feb. 15, 2001, hereby incorporated by referencein its entirety.

SP₃/Edg3 Rat

WO 01/27137, published Apr. 19, 2001, hereby incorporated by referencein its entirety.

S1P₂/Edg5 Human

An, S., Y. Zheng, T. Bleu 2000 Sphingosine 1-Phosphate-induced cellproliferation, survival, and related signaling events mediated by GProtein-coupled receptors Edg3 and Edg5. J. Biol. Chem 275: 288-296,hereby incorporated by reference in its entirety.

WO 99/35259, published Jul. 15, 1999, hereby incorporated by referencein its entirety.

WO99/54351, published Oct. 28, 1999, hereby incorporated by reference inits entirety.

WO 00/56135, published Sep. 28, 2000, hereby incorporated by referencein its entirety.

S1P₂/Edg5 Mouse

WO 00/60056, published Oct. 12, 2000, hereby incorporated by referencein its entirety.

S1P₂/Edg5 Rat

Okazaki, H., N. Ishizaka, T. Sakurai, K. Kurokawa, K. Goto, M. Kumada,Y. Takuwa 1993 Molecular cloning of a novel putative G protein-coupledreceptor expressed in the cardiovascular system. Biochem. Biophys. Res.Comm. 190:1104-1109, hereby incorporated by reference in its entirety.

MacLennan, A. J., C. S. Browe, A. A. Gaskin, D. C. Lado, G. Shaw 1994Cloning and characterization of a putative G-protein coupled receptorpotentially involved in development. Mol. Cell. Neurosci. 5: 201-209,hereby incorporated by reference in its entirety.

U.S. Pat. No. 5,585,476, granted Dec. 17, 1996, hereby incorporated byreference in its entirety.

U.S. Pat. No. 5,856,443, granted Jan. 5, 1999, hereby incorporated byreference in its entirety.

S1P₄/Edg6 Human

Graler, M. H., G. Bernhardt, M. Lipp 1998 EDG6, a novelG-protein-coupled receptor related to receptors for bioactivelysophospholipids, is specifically expressed in lymphoid tissue.Genomics 53: 164-169, hereby incorporated by reference in its entirety.

WO 98/48016, published Oct. 29, 1998, hereby incorporated by referencein its entirety.

U.S. Pat. No. 5,912,144, granted Jun. 15,1999, hereby incorporated byreference in its entirety.

WO 98/50549, published Nov. 12, 1998, hereby incorporated by referencein its entirety.

U.S. Pat. No. 6,060,272, granted May 9, 2000, hereby incorporated byreference in its entirety.

WO 99/35106, published Jul. 15, 1999, hereby incorporated by referencein its entirety.

WO 00/15784, published Mar. 23, 2000, hereby incorporated by referencein its entirety.

WO 00/14233, published Mar. 16, 2000, hereby incorporated by referencein its entirety.

S1P₄/Edg6 Mouse

WO 00/15784, published Mar. 23, 2000, hereby incorporated by referencein its entirety.

S1P₅/Edg8 Human

Im, D.-S., J. Clemens, T. L. Macdonald, K. R. Lynch 2001Characterization of the human and mouse sphingosine 1-phosphatereceptor, S1P₅ (Edg-8): Structure-Activity relationship of sphingosine1-phosphate receptors. Biochemistry 40:14053-14060, hereby incorporatedby reference in its entirety.

WO 00/11166, published Mar. 2, 2000, hereby incorporated by reference inits entirety.

WO 00/31258, published Jun. 2, 2000, hereby incorporated by reference inits entirety.

WO 01/04139, published Jan. 18, 2001, hereby incorporated by referencein its entirety.

EP 1 090 925, published Apr. 11, 2001, hereby incorporated by referencein its entirety.

S1P₅/Edg8 Rat

Im, D.-S., C. E. Heise, N. Ancellin, B. F. O'Dowd, G.-J. Shei, R. P.Heavens, M. R. Rigby, T. Hla, S. Mandala, G. McAllister, S. R. George,K. R. Lynch 2000 Characterization of a novel sphingosine 1-phosphatereceptor, Edg-8. J. Biol. Chem. 275: 14281-14286, hereby incorporated byreference in its entirety.

WO 01/05829, published Jan. 25, 2001, hereby incorporated by referencein its entirety.

Measurement of Cardiovascular Effects

The effects of compounds of the present invention on cardiovascularparameters can be evaluated by the following procedure:

Adult male rats (approx. 350 g body weight) were instrumented withfemoral arterial and venous catheters for measurement of arterialpressure and intravenous compound administration, respectively. Animalswere anesthetized with Nembutal (55 mg/kg, ip). Blood pressure and heartrate were recorded on the Gould Po-Ne-Mah data acquisition system. Heartrate was derived from the arterial pulse wave. Following an acclimationperiod, a baseline reading was taken (approximately 20 minutes) and thedata averaged. Compound was administered intravenously (either bolusinjection of approximately 5 seconds or infusion of 15 minutesduration), and data were recorded every 1 minute for 60 minutes postcompound administration. Data are calculated as either the peak changein heart rate or mean arterial pressure or are calculated as the areaunder the curve for changes in heart rate or blood pressure versus time.Data are expressed as mean±SEM. A one-tailed Student's paired t-test isused for statistical comparison to baseline values and consideredsignificant at p<0.05.

The S1P effects on the rat cardiovascular system are described inSugiyama, A., N. N. Aye, Y. Yatomi, Y. Ozaki, K. Hashimoto 2000 Effectsof Sphingosine-1-Phosphate, a naturally occurring biologically activelysophospholipid, on the rat cardiovascular system. Jpn. J. Pharmacol.82: 338-342, hereby incorporated by reference in its entirety.

Measurement of Mouse Acute Toxicity

A single mouse is dosed intravenously (tail vein) with 0.1 mL of testcompound dissolved in a non-toxic vehicle and is observed for signs oftoxicity. Severe signs may include death, seizure, paralysis orunconciousness. Milder signs are also noted and may include ataxia,labored breathing, ruffling or reduced activity relative to normal. Uponnoting signs, the dosing solution is diluted in the same vehicle. Thediluted dose is administered in the same fashion to a second mouse andis likewise observed for signs. The process is repeated until a dose isreached that produces no signs. This is considered the estimatedno-effect level. An additional mouse is dosed at this level to confirmthe absence of signs.

Assessment of Lymphopenia

Compounds are administered as described in Measurement of Mouse AcuteToxicity and lymphopenia is assessed in mice at three hours post dose asfollows. After rendering a mouse unconscious by CO₂ to effect, the chestis opened, 0.5 mL of blood is withdrawn via direct cardiac puncture,blood is immediately stabilized with EDTA and hematology is evaluatedusing a clinical hematology autoanalyzer calibrated for performingmurine differential counts (H2000, CARESIDE, Culver City Calif.).Reduction in lymphocytes by test treatment is established by comparisonof hematological parameters of three mice versus three vehicle treatedmice. The dose used for this evaluation is determined by tolerabilityusing a modification of the dilution method above. For this purpose,no-effect is desirable, mild effects are acceptable and severely toxicdoses are serially diluted to levels that produce only mild effects.

1. A compound of Formula I

or a pharmaceutically acceptable salt or hydrate thereof, wherein: Ar isphenyl; A is —CO₂H; n is 2, 3 or 4; each R¹ and R² is independentlyselected from the group consisting of: hydrogen, halo, hydroxy, —CO₂H,C₁₋₆alkyl and phenyl, said C₁₋₆alkyl and phenyl optionally substitutedwith 1-3 halo groups; R³ is selected from the group consisting of:hydrogen and C₁₋₄alkyl, optionally substituted with 1-3 hydroxy or halogroups; each R⁴ is independently selected from the group consisting of:hydroxy, halo, —CO₂H, C₁₋₄alkyl, —S(O)_(k)C₁₋₃alkyl, wherein k is 0, 1or 2, C₁₋₃alkoxy, C₃₋₆ cycloalkoxy, aryl and aralkoxy, the alkylportions of said C₁₋₄alkyl, —S(O)_(k)C₁₋₃alkyl, C₁₋₃alkoxy and C₃₋₆cycloalkoxy optionally substituted with 1-3 halo groups; C is HET,wherein HET is thienyl; B is selected from the group consisting of:C₁₋₆alkyl, C₁₋₅alkoxy, —(C═O)—C₁₋₅alkyl, —(C═O)—O—C₁₋₄alkyl,—(C═O)—N(R⁶)(R⁷)—C₁₋₄alkyl,

phenyl and HET; and R⁶ and R⁷ are independently selected from the groupconsisting of: hydrogen, C₁₋₉alkyl and —(CH₂)_(p)-phenyl, wherein p is 1to 5 and phenyl is optionally substituted with 1-3 substituentsindependently selected from the group consisting of: C₁₋₃alkyl andC₁₋₃alkoxy, each optionally substituted with 1-3 halo groups.
 2. Thecompound according to claim 1 wherein n is
 2. 3. The compound accordingto claim 1 wherein n is
 3. 4. The compound according to claim 2 whereineach R¹ and R² is independently selected from the group consisting of:hydrogen, —CO₂H, hydroxy, halo, C₁₋₃alkyl and phenyl.
 5. The compoundaccording to claim 1 wherein R³ is hydrogen or methyl.
 6. The compoundaccording to claim 1 wherein each R⁴ is independently selected from thegroup consisting of: halo, hydroxy, C₁₋₃alkyl, C₁₋₃alkoxy,C₁₋₃alkylthio, phenyl, benzyloxy and cyclopropyloxy.
 7. The compoundaccording to claim 1 wherein B—C is


8. The compound according to claim 1 wherein Ar is phenyl and the group—B—C is attached to the phenyl ring at the 3- or 4-position.
 9. Acompound of Formula II

or a pharmaceutically acceptable salt or hydrate thereof, wherein thegroup —B—C is attached to the phenyl ring at the 3- or 4-position; n is2, 3 or 4; each R¹ and R² is independently selected from the groupconsisting of: hydrogen, —CO₂H, hydroxy, halo, C₁₋₃alkyl and phenyl,said C₁₋₃alkyl and phenyl optionally substituted with 1-3 halo group; Ais —CO₂H; R³ is hydrogen or methyl; each R⁴ is independently selectedfrom the group consisting of: halo, hydroxy, C₁₋₃alkyl, C₁₋₃alkoxy,C₁₋₃alkylthio, phenyl, benzyloxy and cyclopropyloxy; and B—C is


10. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt of any of the foregoing compounds.